GATA4 is diagnostically useful for distinguishing primary ovarian mucinous carcinomas from metastatic colorectal adenocarcinomas to the ovary.

AIM: Determining the primary origin of an ovarian mucin-producing carcinoma can be challenging at times because some metastases of primary colorectal origin may exhibit gross, microscopic, and/or immunohistochemical features that overlap with those of primary ovarian mucinous carcinomas (OMCs). We hypothesized that GATA binding protein 4 (GATA4) might be a novel, useful marker for differentiating primary OMCs from metastatic colorectal adenocarcinomas to the ovary. METHODOLOGY: For comparison with the usefulness of other markers (special AT-rich sequence-binding protein 2 (SATB2) and caudal type homeobox 2 (CDX2)), we elucidated the expression profiles of GATA4 in OMCs, colorectal non-mucinous adenocarcinomas (CNMACs), and colorectal mucinous adenocarcinomas (CMACs) using immunohistochemistry. RESULTS: We confirmed GATA4 expression (H-score ≥50 points) in 93%, SATB2 in 0%, and CDX2 in 64% of 14 OMCs. GATA4 was expressed in 13%, SATB2 in 90%, and CDX2 in 93% of 30 CNMACs. GATA4 was expressed in 20%, SATB2 in 73%, and CDX2 in 100% of 30 CMACs. CONCLUSION: The expression of GATA4 in a mucus-producing ovarian tumor strongly supports it being a primary OMC rather than a metastatic colorectal carcinoma: GATA4 expression indicates OMC and SATB2 expression indicates colorectal adenocarcinoma. However, three cases of colorectal adenocarcinoma were GATA4-positive and SATB2-negative, so the GATA4/SATB2 marker combination is not absolute for determining the primary site. Further research for more markers is necessary to find the ideal combination.

Transdifferentiation of mantle cell lymphoma into sarcoma with limited neuromuscular differentiation after conventional chemotherapy.

We report an exceptionally rare case of mantle cell lymphoma (MCL) that transdifferentiated into sarcoma with limited neuromuscular differentiation. An 81-year-old man with t(11;14)-positive MCL was treated with rituximab and bendamustine and achieved complete remission; however, just 2 months later, the patient developed multiple systemic tumors. Pathologic studies revealed round cell sarcoma expressing synaptophysin, CD56, and myogenin without any B-cell markers. The CCND1 translocation and an identical IGL gene rearrangement were shared by both the MCL and sarcoma. Whole-exome sequencing detected 189 single nucleotide variants (SNVs) in the MCL and 205 SNVs in the sarcoma; 160 SNVs including NSD2, ATM, RB1, and TP53 mutations were shared between MCL and sarcoma cells. An additional PTPN11 mutation was specifically found in the sarcoma. These findings confirmed the shared clonal origin of MCL and sarcoma in this patient and indicated that MCL can transdifferentiate into sarcoma in rare cases.

Expression of CXCL12 in esophageal high grade dysplasia characterized pathologically by lymphocyte accumulation directly under the lesion.

Squamous dysplasia of the esophagus is an unequivocal neoplastic alteration of the esophageal squamous epithelium without invasion. Esophageal high grade dysplasia (EHGD) is characterized by >50% epithelial involvement or severe cytological atypia. Frequently, lymphocytes accumulate below EHGD lesions even though there is no invasion. If this lymphocytic accumulation is active, a transmitter should exist between the EHGD cells and the lymphocytes. C-X-C motif chemokine ligand (CXCL) 12, CXCL10 and C-C motif chemokine ligand 18 (CCL18) are all lymphocyte chemoattractants in vivo, but there are no reports on the relationship between these chemokines and EHGDs. In this study, we investigated these chemokines and C-X-C motif chemokine receptor 4 (CXCR4) (receptor for CXCL12) in 30 EHGDs using immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). For comparison, we enrolled 30 samples of normal esophageal squamous epithelium (NESE). We confirmed CXCL12 expression (H-score≥50 points) in 70% of EHGD and 0% of NESE samples, CXCL10 expression in 3% of EHGD and 3% of NESE samples, CCL18 expression in 3% of EHGD and 0% of NESE samples, and CXCR4 expression in 53% of EHGD and 0% of NESE samples by immunohistochemistry. EHGD and NESE cases were significantly different in their expressions between the tissue types (CXCL12, p<0.001; CXCR4, p<0.001). We examined CXCL12 and CXCR4 mRNA expressions of 3 representative EHGD samples, each having their respective immunostained areas detected by RT-PCR. Finding CXCL12 expression may indicate that this chemokine plays a part in the lymphocyte accumulation that occurs directly under EHGDs.

Expressions of CXCL12, CXCL10 and CCL18 in Warthin tumors characterized pathologically by having a lymphoid stroma with germinal centers.

The Warthin tumor is a benign neoplasm of the salivary glands, histologically, the tumor has an oncocytic epithelial component forming uniform rows of cells surrounded by cystic spaces associated with a lymphoid stroma often showing the presence of germinal centers. The lymphoid stroma is a representative microscopic finding. If this lymphocytic accumulation is active, some sort of transmitter should exist between the Warthin tumor cells and lymphocytes. C-X-C motif chemokine ligand (CXCL12) 12, CXCL10 and C-C motif chemokine ligand 18 (CCL18) are a chemoattractant for lymphocytes in vivo. There is no report on the relationship between these chemokines and Warthin tumors. In this study, we investigated these chemokines expressions in 20 Warthin tumors using immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). For comparison, we also enrolled samples of pleomorphic adenoma, which is another benign salivary gland tumor type without prominent lymphocytic infiltration. All Warthin tumors were immunopositive for CXCL12 and CXCL10, and these reactivities were diffuse. Meanwhile, the majority of pleomorphic adenomas were immunonegative for CXCL12 (95%), CXCL10 (80%) and CCL18 (85%). Warthin tumor and pleomorphic adenoma cases were significantly different in these immunostaining expressions (CXCL12, p<0.001; CXCL10, p<0.001; CCL18, p=0.024). We examined CXCL12, CXCL10 and CCL18 mRNA expressions of 3 representative Warthin tumor samples, each having these chemokines immunopositive areas detected by RT-PCR. Finding CXCL12 and CXCL10 expressions indicate that these chemokines may play a part in the formation of a lymphoid stroma within Warthin tumors. In regards to this phenomenon, the participation of CCL18 might be restrictive compared to CXCL12 and CXCL10.

Significant therapeutic effectiveness of durvalumab after chemoradiotherapy for a patient with post-operative recurrent pulmonary pleomorphic carcinoma.

Pulmonary pleomorphic carcinoma (PPC) is a poorly differentiated non-small cell lung cancer. Because of its rarity, no standard therapy has been established for advanced disease. We herein report on a 62-year-old man with recurrent post-operative PPC, for whom durvalumab after chemoradiotherapy was effective. He was referred to our hospital because of an abnormal shadow in the right upper lung on chest X-ray. After surgical resection was performed, the imaging and histopathological findings revealed PPC (T4N0M0, stage IIIA) with elevated expression of programmed cell death-ligand 1 (PD-L1). A metastasis was found in the left hemithorax 22 months later, and chemoradiotherapy consisting of 60 Gy of radiation and cisplatin plus tegafur/gimeracil/oteracil potassium was administered. Durvalumab was then begun as consolidation therapy. The efficacy of the treatments has continued for longer than 10 months. This case suggests that multidisciplinary treatment with chemoradiotherapy and consolidation immunotherapy may improve the prognosis of locally advanced PPC.

Expressions of IL-8 and CXCL5 in uterine endometrioid carcinomas which have frequent neutrophil infiltration and comparison to colorectal adenocarcinoma.

In endometrioid carcinomas (ECs) of the uterine corpus, neutrophil accumulation within the carcinoma cell clusters is a representative microscopic finding. Because this accumulation is active, some sort of transmitter ought to exist between the EC cells and neutrophils. Interleukin-8 (IL-8) and C-X-C motif chemokine ligand 5 (CXCL5) is a cytokine that attracts neutrophils in vivo. In this study, we investigated IL-8, CXCL5 and C-X-C motif chemokine receptor 2 (CXCR2) (their chemokine receptor) expressions in ECs by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). There are few reports on the relationship between these chemokines and ECs. For comparison, we enrolled samples of colorectal adenocarcinoma (CRAC), it is another representative tumor with neutrophil infiltration. We analyzed 30 ECs and 30 CRACs. We confirmed IL-8 expression (H-score ≥50 points) in 40% of EC and 7% of CRAC samples; CXCL5 expression in 7% of EC and 10% of CRAC samples; CXCR2 expression in 83% of EC and 53% of CRAC samples by immunohistochemistry. We examined each mRNA (IL-8 and CXCL5) expression of 3 representative EC and 3 CRAC samples. Finding IL-8 expression might indicate that this cytokine is important for the process of neutrophil accumulation, particularly within ECs. The participation of CXCL5 regarding neutrophil accumulation within their carcinoma cell clusters might be restrictive compared to IL-8.

Bone Marrow Invasion of Small Cell Neuroendocrine Carcinoma of the Endometrium: A Diagnostic Pitfall Mimicking a Haematological Malignancy.

Metastasis of cancer cells to the bone marrow is relatively rare, despite being one of the most important causes of myelosuppression in patients with solid tumours. A bone marrow examination via a biopsy is the standard method of diagnosing cancer cell invasion into the bone marrow. However, it is sometimes challenging to distinguish neuroendocrine carcinoma cells from haematopoietic cells due to their small, round shape and chromosomal abnormalities resembling haematological malignancies. We herein report a case of bone marrow invasion of small cell neuroendocrine carcinoma of the endometrium mimicking therapy-related myeloid malignancy.

SMARCB1/INI1 Is Diagnostically Useful in Distinguishing α-Fetoprotein-producing Gastric Carcinoma from Hepatocellular Carcinoma.

BACKGROUND/AIM: Switch/sucrose non-fermentable (SWI/SNF)-related, matrix-associated, actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1), also named integrase interactor 1, is one of the core subunit proteins in the SWI/SNF ATP-dependent chromatin remodeling complex encoded at chromosomal position 22q11.2. Complete loss of SMARCB1 expression has been reported in various malignant tumors. Immunohistochemistry has demonstrated that SMARCB1 mutation/inactivation correlates well with loss of nuclear SMARCB1 expression. This study investigated SMARCB1 expression in hepatocellular carcinomas (HCCs) and α-fetoprotein (AFP)-producing gastric carcinomas by immunohistochemistry. For comparison, SMARCB1 immunostaining in intrahepatic cholangiocarcinoma (ICC) was also performed. MATERIALS AND METHODS: Thirty classical HCCs, 30 ICCs and 10 AFP-producing gastric carcinomas were analyzed. RESULTS: Only one case of HCC had focal labeling of SMARCB1 in the nuclei. Twelve cases of ICC were immunopositive for SMARCB1, with either focal or diffuse reactivity. All AFP-producing gastric carcinomas were immunopositive for SMARCB1 in the nuclei, and the reactivity was consistently diffuse. CONCLUSION: SMARCB1 is a potential marker for distinguishing metastatic AFP-producing gastric carcinoma from HCC.

Diagnostic Utility of Prostein, Uroplakin II and SATB2 for Diagnosing Carcinoma of Unknown Primary Origin: A Systematic Immunohistochemical Profiling.

BACKGROUND/AIM: Immunohistochemistry was used to evaluate 600 carcinomas of major histological types from various organs to determine the tissue distributions of the novel markers prostein, uroplakin II and SATB2. MATERIALS AND METHODS: We retrieved 30 cases from 20 different carcinomas of systemic organs. RESULTS: All prostate adenocarcinomas were immunopositive for prostein, and its reactivity was consistently diffuse. There was faint labeling of prostein in few cases of the 570 non-prostatic carcinomas. Uroplakin II was immunopositive in 53% and 60% of urothelial carcinomas (UC) of the bladder and the ureter, respectively. There was focal and weak positivity of uroplakin II in a few cases of non-urinary tract carcinomas. SATB2 was frequently positive in adenocarcinomas of the digestive organs, and was also expressed in a minority of the non-colorectal adenocarcinomas. CONCLUSION: Prostein and uroplakin II are immunohistochemical biomarkers of prostate adenocarcinomas and UCs of the urinary tract.

Single-stranded DNA (ssDNA) Labeling Index Is Related to Risk Grade in Patients with Gastrointestinal Stromal Tumors.

BACKGROUND/AIM: The main objective of the present study was to evaluate the significance of apoptosis in the Fletcher's risk classification for gastrointestinal stromal tumors (GISTs). MATERIALS AND METHODS: Apoptotic cells were identified by immunostaining for single-stranded DNA (ssDNA). We assigned each GIST to one of four risk groups: very low risk, n=32; low risk, n=53; intermediate risk, n=15; high risk, n=6). RESULTS: The mean ssDNA labeling index for each group was 8.0±44.2, 20.1±86.5, 18.7±38.6 and 5.7±5.7, respectively. Fletcher's risk classification for GISTs correlated significantly with the ssDNA labeling index (p=0.002). CONCLUSION: The ssDNA labeling index and Ki-67 labeling index were the most significant factors corresponding to the risk grade of GISTs. These findings suggest that the ssDNA labeling index might be useful for predicting aggressive biological behavior of GISTs.

High expression of CD10 in anaplastic thyroid carcinomas.

AIMS: CD10 is an endopeptidase that degrades various bioactive peptides in the extracellular matrix. In addition to enzymatic degradation, it affects multiple intracellular signal transduction pathways. CD10 expression has been extensively studied in human epithelial cancers of numerous organs and sites. However, its presence in thyroid carcinomas, especially in anaplastic thyroid carcinoma (ATC), has not been fully determined. An actual CD10 expression in thyroid lesions including a large series of ATC was evaluated. METHODS AND RESULTS: We examined CD10 by immunohistochemistry (IHC) in 152 thyroid lesions: nine adenomatous goitres (AGs) and 143 tumours, including 47 anaplastic carcinomas. IHC showed diffuse and strong positivity for CD10 in the epithelial components of almost all ATCs. However, epithelia with squamous metaplasia and oncocytic change from AGs, follicular adenomas and differentiated carcinomas had focal CD10 reactivity. Some papillary thyroid carcinomas (PTCs), along with the PTC components of some ATCs, showed CD10 positivity in fibroblast-like stromal cells and fibrous material. CONCLUSION: Our results imply that the CD10 expression pattern depended on the histotypes of thyroid lesions. When possible metastatic tumours and non-epithelial tumours are excluded, high CD10 expression may be useful in determining whether a primary thyroid carcinoma includes an anaplastic component.

Esophagogastric Junction Carcinomas - Discriminating Histological Types Through Immunohistochemistry.

BACKGROUND/AIM: Although differentiating squamous cell carcinoma (SCC) from adenocarcinoma (AC) at the esophagogastric junction (EGJ) is important for the choice of treatment, this can occasionally be difficult with small biopsy specimens. Therefore, the purpose of this study was to determine the most useful immunomarker panel for discriminating between SCC and AC of the EGJ. MATERIALS AND METHODS: We analyzed 15 SCCs and 26 ACs of the EGJ obtained surgically using immunohistochemistry. RESULTS: The sensitivities of p40, p63 and cytokeratin 5/6 were 100% with specificities of 88%, 46% and 81%, respectively, for SCC. The sensitivities of CAM5.2, caudal-type homeobox 2 (CDX2), mucin-5AC (MUC-5AC) and MUC-6 were 100%, 81%, 77% and 85% with specificities of 27%, 100%, 87% and 87% for AC. CONCLUSION: We demonstrated that a two-marker panel of p40 and CDX2 is highly sensitive and specific.

Frequent BRAF V600E and Absence of TERT Promoter Mutations Characterize Sporadic Pediatric Papillary Thyroid Carcinomas in Japan.

Pediatric papillary thyroid carcinoma (PTC) has unique features but requires further genetic investigation. Moreover, there has been increasing concern about the risk for pediatric PTC in Japan after the Fukushima accident. This study aims to evaluate the frequencies of BRAF and TERT promoter mutations and to examine their significance in non-radiation-associated pediatric PTCs in Japan. We enrolled 81 pediatric PTC patients aged ≤20 years. The control group included 91 adult PTCs from patients >20 years old. BRAF and TERT mutations were analyzed by allele-specific-PCR and/or Sanger sequencing. Compared with adult PTCs, pediatric PTCs exhibited larger tumor size, more frequent lymph node metastasis, and less classical histology. The prevalence of BRAF V600E in pediatric PTCs was 54% and significantly lower than that in adults of 85%. In the pediatric PTCs, BRAF V600E was positively associated with older age, classical histology, and the lymph node metastasis but independent from other clinicopathological factors. TERT mutations were identified in 13% of adults and in none of the pediatric PTCs. In conclusion, pediatric PTCs are characterized by more advanced clinicopathological features, lower BRAF V600E frequency, and absence of TERT mutation. The BRAF V600E frequency in this study is similar to the reported BRAF V600E frequency in the ultrasonographically screened pediatric PTCs in Fukushima.

Genetic alterations of differentiated thyroid carcinoma in iodine-rich and iodine-deficient countries.

BRAF V600E mutation, RET rearrangements, and RAS mutations are the common genetic alterations in differentiated thyroid carcinomas derived from follicular thyroid cells. However, the relationship between these alterations and iodine intake is still controversial. To clarify the influence of iodine intake on the occurrence of differentiated thyroid carcinomas, we performed molecular analyses for two differentiated carcinomas, papillary thyroid carcinomas (PTCs) and follicular thyroid carcinomas (FTCs), from an iodine-rich country (Japan) and an iodine-deficient country (Vietnam). We examined 120 PTCs (67 Japanese and 53 Vietnamese) and 74 FTCs (51 Japanese and 23 Vietnamese). We carried out allele-specific polymerase chain reaction (AS-PCR) for BRAF V600E, PCR and direct sequencing for RAS mutations (codon 12, 13, and 61 in NRAS, HRAS, and KRAS), and RT-PCR for RET/PTC1 and RET/PTC3. BRAF V600E was present in 55/67 (82.1%) Japanese PTCs and 44/53 (83%) Vietnamese PTCs. RET/PTC1 was identified in only one PTC from each country, and no samples had RET/PTC3. NRAS mutation was found in 17/51 (33.3%) Japanese FTCs and 4/23 (17.4%) Vietnamese FTCs. NRAS mutation was cited in codon 61 (20 cases) and codon 12 (one case). None of FTCs had KRAS or HRAS mutations. There were no significant differences in the prevalence of BRAF V600E, RET/PTC, or RAS mutations between the two countries. Our study showed no differences in genetic alterations of thyroid cancers from iodine-rich and iodine-deficient countries, possibly suggesting that iodine intake might not affect the genetic alterations of differentiated thyroid cancer.

Immunohistochemical detection of NRAS(Q61R) protein in follicular-patterned thyroid tumors.

The NRAS(A182G) mutation, which results in the NRAS(Q61R) protein, is a major driver mutation in follicular-patterned thyroid neoplasms. Although new immunohistochemistry (IHC) for NRAS(Q61R) is now available, its sensitivity, specificity, and diagnostic utility for thyroid tumors are not yet established. We performed IHC for NRAS(Q61R) and direct sequencing for NRAS codon 61 in 4 thyroid cancer-derived cell lines and 98 follicular-patterned thyroid tumors that included 22 follicular thyroid adenomas (FTAs), 35 follicular thyroid carcinomas (FTCs), and 41 cases of nodular hyperplasia (NH). In the tumors with NRAS(Q61R), the expression of BRAF(V600E) was further evaluated immunohistochemically. Two cell lines with NRAS(A182G) showed selective immunoreactivity for NRAS(Q61R). In tumor tissues, NRAS(Q61R) IHC was positive in 18% (4/22), 29% (10/35), and 2% (1/41) of FTAs, FTCs, and NH samples, respectively. The frequencies of the NRAS(Q61R) in FTAs and FTCs were significantly higher than that in NH (P=.046 and P=.001, respectively). All tumors with NRAS(Q61R) expression exhibited uniform cytoplasmic positivity with or without accumulation in their cell membranes. Of the 15 tumors with NRAS(Q61R) expression, 13 cases showed NRAS(A182G) in direct sequencing, whereas all of the tumors without NRAS(Q61R) expression were negative for the mutation. There were no tumors with overlapping expression of NRAS(Q61R) and BRAF(V600E). In reference to the direct sequencing, sensitivity and specificity of the NRAS(Q61R) IHC were 100% and 98%, respectively. In conclusion, NRAS(Q61R) IHC is a highly sensitive and specific tool that is useful for differentiating follicular-patterned thyroid tumors.

Spindle cell oncocytoma of adenohypophysis: Report of a case and immunohistochemical review of literature.

We present a case of spindle cell oncocytoma (SCO) of the adenohypophysis in a 70-year-old Vietnamese male. The patient was admitted to Cho Ray Hospital after suffering from headache and visual disturbance for 6 months. Clinicians detected a 60×55×45 mm(3) mass located in the suprasellar-sellar region. Histopathologically, the resected tumor was composed of spindle cells with oncocytic appearance. Immunohistochemical examination revealed expression of anti-mitochondria antibody (AMA), vimentin, thyroid transcription factor 1 (TTF-1), epithelial membrane antigen (EMA) and galectin-3. These histologic and immunohistochemical findings are suggestive of SCO.

Branchial Cleft-Like Cysts Involving 3 Different Organs: Thyroid Gland, Thymus, and Parotid Gland.

Branchial cleft cysts (BCCs) are also named lateral cervical cysts and widely acknowledged as being derived from embryonic remnants. Lymphoepithelial cysts (LECs) generally show microscopic features that are identical to those of BCCs, and rarely occur at unusual sites or organs.A case of multiple cysts arising in both lobes of the thyroid gland, thymus, and right parotid gland in a 41-year-old man is reported. Clinically, the patient presented with Hashimoto's thyroiditis for about 20 years and had past histories of idiopathic thrombocytopenic purpura and severe respiratory infection.This case is unusual in that multiple cysts arose synchronously and/or heterochronously and grew, increasing their sizes in these different organs. Microscopic examinations revealed that all of the cysts were composed of squamous epithelium, dense lymphoid tissue with germinal centers, and a fibrous capsule. These findings corresponded to those of BCCs or LECs. It is notable that the histopathological features were nearly the same in the individual organs. A review of the literature disclosed no previous such reported cases.The etiology is unknown. However, based upon the similar histopathological features of all the excised specimens, common immune and/or hematopoietic disorders may have contributed to their occurrence and development in association with putative genetic abnormalities.

Signet ring cell carcinoma of the non-ampullary duodenum: A case report.

We describe a case of signet ring cell carcinoma of the non-ampullary duodenum in an 86-year-old woman. Endoscopic examination revealed a fungating lesion (Borrmann classification; type 2) on the anterior wall of the descending duodenum (second part). Pylorus-preserving pancreaticoduodenectomy and lymphadenectomy were performed. The tumor (pT3 and pN0) was predominantly composed of signet ring cells, characterized by a central, optically clear, globoid droplet of cytoplasmic mucin with an eccentrically placed nucleus. The cells were positive for periodic acid Schiff with diastase and Alcian blue pH 2.5. On immunohistochemical examination, MUC-5AC, MUC-2 and CDX2 were diffusely positive, and MUC-6 was negative in the tumor cells, which consisted of mixed gastric foveolar and intestinal phenotypes. Cytokeratin 7 was focally positive, but cytokeratin 20 was negative. Nuclear staining of p53 was diffusely and weakly positive. Signet ring cell carcinoma occurring in the non-ampullary duodenum might derive from duodenal goblet cells with MUC-5AC expression.

Squamous morula formation in colorectal adenoma: Immunohistochemical and molecular analyses.

Little is known about the squamous morular component (SMC) in colorectal neoplasms because of its rarity. The aim of the present study is to elucidate the morphological, immunohistochemical and genetic characteristics of SMCs in colorectal adenomas. Five colorectal adenomas having SMCs were resected from five patients endoscopically. On immunohistochemical examination (four cases), all SMCs were positive for cytokeratin 5/6 in their cytoplasm and positive for ß-catenin in their cytoplasm and nuclei. A nuclear positivity of p63 was detected in one SMC. All SMCs were negative for p53, chromogranin A, synaptophysin and NCAM. There was no Ki-67 expression in any of the SMCs. We detected none of mutations of ß-catenin, KRAS and BRAF by microdissection and polymerase chain reaction-direct sequence in any of the four examined SMCs. SMCs are a rare but problematic finding in colorectal adenomas. Using immunohistochemistry for ß-catenin, cytokeratin 5/6, Ki-67, p53, chromogranin A, synaptophysin and NCAM can facilitate the diagnosis of these peculiar cell nests.