Expanding the spectrum of tyrosine kinase fusions in calcified chondroid mesenchymal neoplasms: Identification of a novel PDGFRA::USP8 gene fusion.

Calcified chondroid mesenchymal neoplasms represent a distinct, and recently recognized, spectrum of tumors. To date most cases have been reported to be characterized by FN1 gene fusions involving multiple potential tyrosine kinase partners. Following incidental identification of a tumor morphologically corresponding to calcified chondroid mesenchymal neoplasm, but with a PDGFRA::USP8 gene fusion, we undertook a retrospective review to identify and characterize additional such cases. A total of four tumors were identified. Each was multilobulated and composed of polygonal-epithelioid-stellate cells with a background of chondroid matrix containing distinctive patterns of calcification. Targeted RNA sequencing revealed an identical PDGFRA (exon 22)::USP8 (exon 5) gene fusion in each case. Subsequent immunohistochemical staining confirmed the presence of PDGFRα overexpression. In summary, we report a series of four tumors within the morphologic spectrum of calcified chondroid mesenchymal neoplasms. In contrast to prior reports, these tumors harbored a novel PDGFRA::USP8 gene fusion, rather than FN1 rearrangement. Our findings expand the molecular diversity of these neoplasms, and suggest they are united through activation of protein kinases.

Spatial Distribution and Densities of CD103+ and FoxP3+ Tumor Infiltrating Lymphocytes by Digital Analysis for Outcome Prediction in Breast Cancer.

BACKGROUND: The evaluation of tumor-infiltrating lymphocytes (TILs) for breast cancer prognosis is now established. However, the clinical value for their spatial distributions of specific immune subsets, namely CD103+ tissue-resident memory T cells FoxP3+ regulatory T ells, have not been thoroughly examined. METHOD: Representative whole sections of breast cancers were subjected to CD103 and FoxP3 double staining. Their density, ratio, and spatial features were analyzed in tumor area and tumor-stromal interface. Their associations with clinicopathological parameters and patient's prognosis were analyzed. RESULTS: CD103 TILs were closer to tumor nests than FoxP3 TILs in the tumor-stromal interface. Their densities were associated with high-grade disease, TNBC, and stromal TILs. High stromal FoxP3 (sFoxP3) TILs and close proximity of sCD103 TILs to tumor were independently associated with better survival at multivariate analysis. Subgroup analysis showed the high FoxP3 TILs density associated better survival was seen in HER2-OE and TNBC subtypes while the proximity of CD103 TILs to tumor nests associated better survival was seen in luminal cancers. CONCLUSION: The prognostic impact of CD103 and FoxP3 TILs in breast cancer depends on their spatial localization. High sFoxP3 TIL density and the lower distance of CD103 TILs from the tumor nests had independent favorable prognostic values.

Colorectal Carcinoma With Sarcomatoid Components: Report of 15 Cases and Literature Review of an Exceedingly Rare Carcinoma Subtype.

Colorectal carcinoma with sarcomatoid components (which includes so-called carcinosarcomas and sarcomatoid carcinomas) is a rare subtype with 50 reported cases in the literature and overlapping criteria with undifferentiated carcinoma. We collected and described 15 cases from 10 men and 5 women, with a mean age of 66 years. Symptoms included abdominal pain and gastrointestinal bleeding. Most tumors presented in the rectosigmoid region, with a mean size of 8.2 cm. The sarcomatoid component, on average, represented 58% of the tumors and took many forms, including spindled (10 cases), anaplastic (9 cases), and rhabdoid (3 cases); one case showed osteoid matrix. Tumor budding was usually high, and tumor-infiltrating lymphocytes were usually low. The sarcomatoid component was keratin-positive in 10 cases. One case showed loss of mismatch repair protein expression, and 2 cases showed SMARCA4 loss (1 also with SMARCA2 loss). Molecular testing identified mutations in KRAS (n=1), NRAS (n=2), BRAF (n=2), APC (n=1), and TP53 (n=1) in a few cases. Tumors often presented at advanced stage, with 11 cases pT4, 9 cases with nodal metastases, and 7 cases with distant metastases. Follow-up was available for 10 cases (median: 2 months), with 2 alive without disease, 3 alive with disease, and 5 dead. Our findings roughly corresponded with those in previously reported cases. Colorectal carcinoma with sarcomatoid components is rare and aggressive, with a poor prognosis for many patients. We suggest that spindled cells, anaplasia, heterologous elements, and/or a component with definable sarcomatous lineage be used to distinguish colorectal carcinoma with sarcomatoid components from undifferentiated carcinoma.

Gorham-Stout disease: remission with sirolimus therapy.

Gorham-Stout disease (GSD) is a rare, non-hereditary, bone disease characterised by progressive osteolysis as a result of uncontrolled proliferation of endothelial-lined vessels replacing normal bone. We present a baby-girl with the classic radiological features of GSD and compatible clinical and histological findings, who developed progressive disease for over 2 years despite propranolol treatment. Propranolol treatment was stopped and sirolimus monotherapy started which resulted in near-complete resolution after 1 year, with no recurrence after discontinuation of treatment. This case not only illustrates the typical features of GSD on a variety of imaging modalities, but is also the first report showing stark contrast in response between propranolol and sirolimus treatment for GSD, highlighting how targeting lymphatic, rather than solely angiomatous, proliferation at the vascular endothelial growth factor-level may be a future direction.

Validation of a novel risk score to predict early and late recurrence in solitary fibrous tumour.

BACKGROUND: Current risk models in solitary fibrous tumour (SFT) were developed using cohorts with short follow-up and cannot reliably identify low-risk patients. We recently developed a novel risk model (G-score) to account for both early and late recurrences. Here, we aimed to validate the G-score in a large international cohort with long-term follow-up. METHODS: Data were collected from nine sarcoma referral centres worldwide. Recurrence-free interval (RFi) was the primary endpoint. RESULTS: The cohort comprised 318 patients with localised extrameningeal SFTs. Disease recurrence occurred in 96 patients (33%). The estimated 5-year RFi rate was 72%, and the 10-year RFi rate was 52%. G-score precisely predicted recurrence risk with estimated 10-year RFi rate of 84% in low risk, 54% in intermediate risk and 36% in high risk (p < 0.001; C-index 0.691). The mDemicco (p < 0.001; C-index 0.749) and SalasOS (p < 0.001; C-index 0.674) models also predicted RFi but identified low-risk patients less accurate with 10-year RFi rates of 72% and 70%, respectively. CONCLUSIONS: G-score is a highly significant predictor of early and late recurrence in SFT and is superior to other models to predict patients at low risk of relapse. A less intensive follow-up schedule could be considered for patients at low recurrence risk according to G-score.

Current Developments of Artificial Intelligence in Digital Pathology and Its Future Clinical Applications in Gastrointestinal Cancers.

The implementation of DP will revolutionize current practice by providing pathologists with additional tools and algorithms to improve workflow. Furthermore, DP will open up opportunities for development of AI-based tools for more precise and reproducible diagnosis through computational pathology. One of the key features of AI is its capability to generate perceptions and recognize patterns beyond the human senses. Thus, the incorporation of AI into DP can reveal additional morphological features and information. At the current rate of AI development and adoption of DP, the interest in computational pathology is expected to rise in tandem. There have already been promising developments related to AI-based solutions in prostate cancer detection; however, in the GI tract, development of more sophisticated algorithms is required to facilitate histological assessment of GI specimens for early and accurate diagnosis. In this review, we aim to provide an overview of the current histological practices in AP laboratories with respect to challenges faced in image preprocessing, present the existing AI-based algorithms, discuss their limitations and present clinical insight with respect to the application of AI in early detection and diagnosis of GI cancer.

Development of deep learning algorithms to discriminate giant cell tumors of bone from adjacent normal tissues by confocal Raman spectroscopy.

Raman spectroscopy is a non-destructive analysis technique that provides detailed information about the chemical structure of tumors. Raman spectra of 52 giant cell tumors of bone (GCTB) and 21 adjacent normal tissues of formalin-fixed paraffin embedded (FFPE) and frozen specimens were obtained using a confocal Raman spectrometer and analyzed with machine learning and deep learning algorithms. We discovered characteristic Raman shifts in the GCTB specimens. They were assigned to phenylalanine and tyrosine. Based on the spectroscopic data, classification algorithms including support vector machine, k-nearest neighbors and long short-term memory (LSTM) were successfully applied to discriminate GCTB from adjacent normal tissues of both the FFPE and frozen specimens, with the accuracy ranging from 82.8% to 94.5%. Importantly, our LSTM algorithm showed the best performance in the discrimination of the frozen specimens, with a sensitivity and specificity of 93.9% and 95.1% respectively, and the AUC was 0.97. The results of our study suggest that confocal Raman spectroscopy accomplished by the LSTM network could non-destructively evaluate a tumor margin by its inherent biochemical specificity which may allow intraoperative assessment of the adequacy of tumor clearance.

Expanding the spectrum of mesenchymal neoplasms with NR1D1-rearrangement.

Undifferentiated mesenchymal neoplasms can be morphologically subclassified based on cell shape; epithelioid tumors may be diagnostically challenging, particularly since they can show morphologic and immunohistochemical overlap with epithelial neoplasms. Following the recent report of an NR1D1::MAML1 gene fusion in an undifferentiated pediatric neoplasm, we performed a retrospective archival review and identified four additional cases of undifferentiated mesenchymal neoplasms with NR1D1-rearrangement. All four tumors occurred in adult women. The tumors involved superficial and/or deep soft tissues of the extremities or abdomen. Morphologically, they showed a spectrum of overlapping features. In addition to epithelioid cells, two cases also had a prominent spindle cell component. Two cases also had admixed polygonal cells containing prominent cytoplasmic vacuoles with amorphous debris. The immunophenotype was nonspecific but all cases had at least focal keratin expression; this was extensive in two tumors. Targeted RNA-sequencing revealed two cases each with NR1D1::MAML1 and NR1D1::MAML2 gene fusions. One patient developed lung and liver metastases, and one patient required amputation due to multifocal disease and underlying bone involvement. This study confirms undifferentiated NR1D1-rearranged sarcoma represents a distinct mesenchymal neoplasm with an epithelioid morphology and potential for aggressive behavior. Further, we offer new insight into the spectrum of clinical, morphologic, immunohistochemical, and molecular findings possible in these rare neoplasms. An awareness of this entity is especially important given the potential for misclassification as a carcinoma.

The novel finding of an FGFR1::TACC1 fusion in an undifferentiated spindle cell sarcoma of soft tissue with aggressive clinical course.

We report an aggressive soft tissue sarcoma with FGFR1-TACC1 fusion occurring in the thigh of an 83-year-old man. Microscopically, the tumor was composed of monomorphic spindle cells arranged in intersecting fascicles. The tumor cells showed ovoid nuclei, fine chromatin, indistinct nucleoli, and elongated eosinophilic cytoplasm. Focal increase in nuclear atypia was noted. Immunohistochemistry showed only focal rare positivity to S100, but negative to SOX10, CD34, STAT6, TLE-1, SMA, and other myogenic markers. An extensive panel of immunostains did not reveal a definite lineage of cellular differentiation. The fusion junction of the chimeric transcript involved FGFR1 exon 16 and TACC1 exon 7, which was similar to those reported in other types of neoplasms such as glioblastomas and epithelial cancers. The transcript was predicted to be in-frame and confirmed by Sanger sequencing after reverse transcriptase-polymerase chain reaction. The patient was treated by marginal excision of tumor without receiving adjuvant therapy. He experienced rapid tumor recurrence with distant metastases and succumbed at 3.5 months after presentation. The finding of FGFR1-TACC1 fusion in a high-grade, undifferentiated spindle cell sarcoma of soft tissue is novel and may offer a potential therapeutic target in the near future.

NKX3-1 Is a Useful Immunohistochemical Marker of EWSR1-NFATC2 Sarcoma and Mesenchymal Chondrosarcoma.

NK3 homeobox 1 (NKX3-1) is widely accepted as a highly sensitive and specific marker for prostatic adenocarcinoma. Prompted by published transcriptome data showing upregulation of NKX3-1 mRNA expression in EWSR1-NFATC2 sarcoma, we explored the utility of NKX3-1 immunohistochemistry in sarcoma diagnosis. We applied NKX3-1 immunohistochemistry to 11 EWSR1-NFATC2 sarcomas and 168 mimics using whole tissue sections. All EWSR1-NFATC2 sarcomas consisted of uniform small round or ovoid cells, all except 1 showing at least focally the typical growth pattern of nests, cords, or trabeculae within a fibrous/myxoid background. A variable eosinophilic infiltrate was common. NKX3-1 was expressed in 9 of 11 (82%) EWSR1-NFATC2 sarcomas, often diffuse and of moderate or strong intensity. All 12 mesenchymal chondrosarcomas tested were also positive for NKX3-1, with over half showing diffuse staining and moderate or strong intensity. The positive staining was seen only in the primitive small round cell component, whereas the cartilaginous component was mostly negative. Although 1 of 30 osteosarcomas showed focal NKX3-1 positivity, all the remaining 155 cases tested, including 20 Ewing sarcomas, 20 myoepithelial tumors, 11 ossifying fibromyxoid tumors, and 1 FUS-NFATC2 sarcoma were negative for NKX3-1. Our study provides the first evidence that EWSR1-NFATC2 sarcoma and Ewing sarcoma could be distinguished immunohistochemically, adding to the accumulating data that these tumors are phenotypically distinct. We suggest that NKX3-1 may have a diagnostic utility in the evaluation of sarcoma and we also call attention to potential pitfalls in the use of this well-known marker of prostatic adenocarcinoma.

Uterine Tumor Resembling Ovarian Sex Cord Tumor (UTROSCT): A Morphologic and Molecular Study of 26 Cases Confirms Recurrent NCOA1-3 Rearrangement.

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare mesenchymal neoplasm, of uncertain biological potential, that was recently reported to exhibit recurrent gene fusions involving NCOA2-3. The purpose of this study was to, using a larger sample size, better characterize the histopathologic and molecular diversity of UTROSCT. Twenty-six cases of UTROSCT from 5 institutions were selected for further study. Fluorescence in situ hybridization for NCOA1, NCOA2, NCOA3, ESR1 and GREB1, and targeted RNA sequencing was performed on 17 and 8 UTROSCTs, respectively. Eight cases underwent massively parallel sequencing to detect single nucleotide variants (SNV), copy number variations, and structural variants using a targeted hybrid-capture based assay. NCOA1-3 rearrangement was identified in 81.8% (18/22) of cases. The most common fusion was ESR1-NCOA3, occurring in 40.9% (9/22). GREB1-NCOA1 (n=4), ESR1-NCOA2 (n=3), and GREB1-NCOA2 (n=1) rearrangements were also identified. No recurrent SNVs were identified and no tumor had SNVs in FOXL2, DICER1, STK11, or AKT1, which can be seen in ovarian sex cord-stromal tumors. Copy number variations were infrequent. Clinical follow-up was available for 11 cases with a mean follow-up interval of 94.4 (range, 1 to 319) months. Only one case had a recurrence 66 months after the initial diagnosis and this was the single case with a GREB1-NCOA2 fusion. This study reports the morphologic spectrum of UTROSCT and confirms the recently reported recurrent NCOA2-3 gene fusions, in addition to identifying novel rearrangements involving NCOA1 in these tumors.

PRRX-NCOA1/2 rearrangement characterizes a distinctive fibroblastic neoplasm.

Fibroblastic/myofibroblastic neoplasms represent a broad, and occasionally diagnostically challenging, category of soft tissue neoplasms. A subset of these tumors defy conventional classification. However, with the advent of next-generation sequencing, the identification of disease-defining molecular alterations is gradually improving their subclassification. Following identification of two index cases of a distinctive fibroblastic neoplasm with a fusion gene involving PRRX1 and NCOA1, we performed a retrospective review to further characterize this entity. We identified two additional cases, including one with a fusion between PRRX1 and NCOA2. The average patient age was 38 years, and three patients were female. Two tumors occurred on the neck, and the others involved the groin and thigh. Tumors were centered in the subcutis and ranged from 2.3 to 14.0 cm (average 5.8 cm). Morphologically, they were predominantly hypocellular, with focal hypercellularity. They were composed of monomorphic spindle-stellate cells with a vague fascicular pattern. The nuclei were bland with only rare mitotic activity, and occasional multinucleation. The intervening stroma was typically abundant and ranged from myxoid to collagenous, with frequent rope-like collagen bundles. Three of the cases had a prominent vasculature ranging from numerous small curvilinear vessels to ectatic and branching staghorn-like vessels. Immunohistochemistry was negative for desmin, smooth muscle actin, S100, CD34, keratin, and epithelial membrane antigen. Each of the patients was treated by simple excision and none of the tumors were associated with local recurrence or metastasis. Based on their unique morphological and molecular attributes, we believe this represents a novel fibroblastic tumor for which we have tentatively proposed the name "PRRX-NCOAx-rearranged fibroblastic tumor."

Association of clinicopathological features and prognosis of TERT alterations in phyllodes tumor of breast.

Phyllodes tumor (PT) of the breast is a rare but clinically important fibroepithelial tumor with potential risks of recurrence and metastasis. Recent studies identified recurrent TERT promoter mutations in PTs. However, the clinical significance of this alteration has not been fully examined. Two hundred and seven PTs from two intuitions were included. All cases were subjected to immunohistochemical analysis for TERT expression. Analysis of TERT promoter mutations was further performed by Sanger sequencing targeting the hotspot mutation region on cases from one of the involved institutions. The expression of TERT was correlated with clinicopathologic features, mutation status and recurrence. There was an association of TERT expression and its promoter mutation. Both stromal TERT expression and its promoter mutation correlated with PT grading and older patient age. Recurrence free survival (RFS) of PT patients with high stromal TERT expression was shorter if the excision margin was positive. Our findings suggested a possible pathogenic role of TERT alteration in PT malignancy. Currently there is no consensus for re-excision for PT patients with positive surgical margin, particularly for low grade cases. Stromal TERT expression could be potentially useful to guide management patients with benign PTs.

Anterior Gradient 2 is a Poor Outcome Indicator in Luminal Breast Cancer.

BACKGROUND: Recent data have shown anterior gradient 2 (AGR2) to be a novel oncogene overexpressed in luminal breast cancers. However, many studies only focused on its relationship with treatment resistance, and the clinical relevance of AGR2 has not been widely evaluated. METHODS: AGR2 expression in a cohort of breast cancer was correlated with the clinicopathologic features, biomarker expression, and patient survival. RESULTS: AGR2 expression correlated with lower grade (p = 0.002) and absence of necrosis (p = 0.001) and was most highly expressed in luminal cancers (p < 0.001). Correspondingly, AGR2 correlated positively with estrogen receptor, progesterone receptor, and androgen receptor (p < 0.001) and negatively with epidermal growth factor receptor (p =0.002) and CK5/6 (p < 0.001). AGR2 was an independent unfavorable disease-free survival prognostic factor in patients with hormone therapy (hazard ratio 2.537, p = 0.023). Interestingly, it was also an independent adverse disease-free survival prognostic factor in node-positive luminal cancers (hazard ratio 3.381, p = 0.016). AGR2 expression was higher in nodal metastasis than the corresponding primary tumors in luminal cancers (p= 0 .023). CONCLUSIONS: The prognostic impact of AGR2 expression could be related to treatment outcome in estrogen receptor-positive breast cancers and/or its metastasis-promoting effects. It could be an important tumor biomarker and negative prognostic factor potentially exploitable in clinical practice.

Immunohistochemistry in the diagnosis of papillary lesions of the breast.

AIMS: Many immunohistochemical markers have been studied for differentiating papillary lesions. However, their differentiating power has not been evaluated comprehensively. Additionally, assessment of some markers will require the difficult task of identifying different cell types. In the current study, we aimed to devise a simple papillary panel which can aid in diagnosis irrespective of architectural pattern and cell type differentiation. METHODS AND RESULTS: Immunohistochemical analysis of papillary lesions using myoepithelial markers [p63 and smooth muscle actin (SMA)], high molecular weight cytokeratins (HMWCKs: CK5, CK5/6, CK14 and 34ßE12), hormone receptors (ER and PR) and neuroendocrine markers (chromogranin and synaptophysin) was performed. Among them, neuroendocrine markers showed high specificity but low sensitivity. HMWCK specificity was better than that for myoepithelial markers. Homogeneous staining pattern for hormonal receptors rather than their percentage positivity was more effective in identifying malignant lesions. Negative staining for two or more of HMWCKs, namely CK5/6, CK14 and 34ßE12, achieved the best overall specificity and sensitivity of 87.8% and 94.1%, respectively, irrespective of the architecture. Their discriminatory power was validated with an independent cohort of core needle biopsies. CONCLUSIONS: A marker panel with HMWCKs could be used in differentiating papillary lesions irrespective of architectural pattern or cell type differentiation.

Columnar cell-like changes in the male breast.

AIMS: Columnar cell lesions are known as a link between normal breast and low grade neoplastic lesions in female, but have not been established in the male breast. This study evaluated the presence of ducts showing columnar cell-like features in the male breast. METHODS: Seventy-one consecutive surgical resections from men (6 invasive breast carcinoma of grade 3, 1 atypical ductal hyperplasia and 64 other lesions) were reviewed to identify foci of dilated ducts with columnar epithelial cells, and their morphological features including apical snouts, intraluminal secretions and calcifications were assessed. The expression of CK5/6 and estrogen receptor (ER) was evaluated immunohistochemically. Clinicopathological features including patients' age, histological diagnosis and gynaecomastoid hyperplasia were documented. RESULTS: Ducts showing columnar cell-like features were identified in 39 cases, morphologically as distended ducts with round or undulating outline. There was an outer layer of myoepithelial cells and an inner layer of columnar luminal cells showing apical snouts, but without intraluminal secretions or calcifications. Immunohistochemically, these columnar epithelial cells were negative for CK5/6 in 38/39 cases and all were ER heterogeneously positive. These changes were associated with older age, but their incidence did not differ whether they were associated with invasive breast carcinoma, atypical ductal hyperplasia and other lesions. CONCLUSIONS: In the male breast, there is an entity sharing morphological features and immunohistochemical profile of columnar cell lesions.

Nerve growth factor receptor (NGFR): a potential marker for specific molecular subtypes of breast cancer.

AIMS: Nerve growth factor receptor (NGFR) is a transmembrane receptor for the neurotrophin family. It acts either as tumour suppressor or oncogene depending on cellular context. Its role in breast cancers remained conflicting, possibly due to the heterogeneity of breast cancer subtypes. METHODS: In this study, we have analysed NGFR expression in 602 cases of breast cancers by immunohistochemistry. Its expression was correlated with biomarker expression and different breast cancer subtypes. RESULTS: NGFR expression was found to be positively correlated with basal markers, including Ki67, Cytokeratin (CK5/6), CK14, p63, c-kit and Epidermal growth factor receptor (EGFR) , but negatively with hormonal receptors. Among different molecular subtypes, it was negatively associated with luminal A, but positively with luminal B, and basal-like breast cancer BLBC subtypes. When comparing NGFR with other basal markers in BLBC, though less sensitive, its specificity was comparable to or better than other basal markers. For luminal B cancers, NGFR showed a high specificity which was also comparable to or better than the defining markers (estrogen receptor (ER), progesterone receptor (PR), Human epidermal growth receptor 2 (HER2) and Ki-67) for the subtype. CONCLUSIONS: Overall, these findings suggested that NGFR expression could be indicative for the BLBCs or luminal B subtypes. It may represent a potential adjunct marker for these two subtypes.

Biopsy sampling of breast lesions: comparison of core needle- and vacuum-assisted breast biopsies.

Needle biopsy is now the initial investigation of choice for the pre-operative diagnosis of breast lesions. This includes core needle biopsy (CNB) and vacuum-assisted biopsy (VAB) with or without radiologic assistance. The performance indices of both of these biopsy techniques were evaluated. In a large cohort of patients with breast lesions including 464 cases (285 CNB and 179 VAB), with confirmed outcomes, the diagnostic accuracy was compared using parameters including quantitation of the sampling based on the total number of cores taken, cores containing breast parenchyma, and cores with lesion; and non-epithelial changes including necrosis and calcification. CNB showed a 99% PPV, 94% NPV, 96% sensitivity, and 99% specificity, whereas VAB demonstrated a 100% PPV, 100% NPV, 100% sensitivity, and 100% specificity. The correct diagnosis in CNB was proportional to the number of cores extracted, whereas accuracy of VAB was independent of the total number of cores taken. There was a positive correlation between the presence of calcification and malignancy in CNB, but not detected under VAB. CNB and VAB were equally efficient in palpable lesions, in detecting necrosis, and calcification. Large calcification was found to be associated with malignancy in both CNB and VAB. In non-palpable lesions, VAB was more effective in the detection of calcification. The diagnostic accuracy of VAB appeared to be independent of number of cores sampled, whereas CNB required a minimum of 3-4 cores to achieve high diagnostic accuracy.