Laser pressure catapulting (LPC): Optimization LPC‐system and genotyping of colorectal carcinomas

Bazan, Viviana; Rocca, Gaspare La; Corsale, S; Agnese, Valentina; Macaluso, Marcella; Migliavacca, Manuela; Gregorio, Valter; Cascio, Sandra; Sisto, Pasqua Sandra; Fede, Gaetana Di; Buscemi, M; Fiorentino, Eugenio; Passantino, Rosa; Morello, Vincenza; Rm, Tomasino; Russo, Antonio

doi:10.1002/jcp.20150

Cited by 21 publications

(13 citation statements)

References 17 publications

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“…Others have found that combining laser microdissection with sensitive molecular methods such as real-time PCR, previously unanswerable questions about the development and progression of human cancer can be explored [24][25][26]. Notwithstanding, in our study, the increased cyclin A2 protein expression could not be explained by CCNA2 amplification analyses in isolated colonic tumour cells.…”

Section: Discussioncontrasting

confidence: 49%

Cyclin A2 Protein Overexpression Is Not Caused by Gene Amplification in Colon Cancer

et al. 2012

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Increased expression of cyclin A2 protein has been detected in different types of cancers. However, its prognostic importance appears to differ between tumours. The significance and precise mechanisms behind cyclin A2 overexpression remain to be elucidated. We used real-time PCR to examine CCNA2 amplification in tumour cells isolated by laser microdissection and in total tumour tissue in colon cancer patients in which overexpression of cyclin A2 protein had been revealed by immunohistochemistry (n = 22 patients). The results were verified by FISH. CCNA2 amplification was not detected in either the isolated tumour cells or the total tumour tissue. We verified our methods by demonstrating amplification of CCNA2 by real-time PCR in three out of eight breast tumours that overexpressed cyclin A2 protein (this frequency is consistent with the findings of others). However, FISH did not reveal any CCNA2 amplification in the breast tumours, but it did reveal polysomy of chromosome 4 or segments of chromosome 4 in three tumour tissue samples, indicating the importance of verifying the real-time PCR results with another method. To conclude, the increased cyclin A2 protein expression in these patients could not be explained by CCNA2 amplification in isolated colonic tumour cells.

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Section: Discussioncontrasting

confidence: 49%

Cyclin A2 Protein Overexpression Is Not Caused by Gene Amplification in Colon Cancer

et al. 2012

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“…Another way to increase tumor cell enrichment is to perform a more selective dissection of the scattered tumor cells/glands by laser capture microdissection. 17,30 In our hands, laser capture microdissection is efficient (two additional mutations were identified), but does not seem to be suitable for a routine setting because it is time-and laborconsuming and, therefore, expensive. In practice, 10-100 min were necessary for microdissecting a sample, depending on its tumor cellularity.…”

Section: Discussionmentioning

confidence: 89%

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

Boissière‐Michot¹,

Lopez-Crapez²,

Frugier³

et al. 2012

Modern Pathology

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KRAS status assessment is mandatory in patients with metastatic colorectal cancer before therapy with antiepidermal growth factor receptor monoclonal antibodies, as KRAS mutations are associated with resistance to this treatment. However, KRAS genotyping may be very challenging in case of poor tumor cellularity, particularly when major tumor regression is achieved in locally advanced rectal adenocarcinomas after radiochemotherapy. We aimed at identifying the most reliable strategy to detect KRAS mutations in such samples. DNA was extracted from 31 surgical specimens with major tumor regression, following manual dissection, and from paired pre-treatment biopsies and analyzed by high-resolution melting. DNA samples displaying altered melting curve shapes were then sequenced. Samples with unmodified melting curves or wild-type sequence were further investigated by using an allele-specific PCR assay (TheraScreen) and laser microdissection (followed by high-resolution melting and sequencing analyses). In the 31 post-radiochemotherapy surgical specimens, seven KRAS mutations were identified by high-resolution melting analysis/sequencing. One additional mutation was detected by the TheraScreen assay and two mutations, including the one identified by the TheraScreen assay, were detected following laser microdissection. Altogether, 9/31 surgical specimens (29%) presented KRAS mutations. In the manually dissected pre-treatment biopsies, 12 mutations (39%) were identified by high-resolution melting analysis and sequencing. No additional mutations were found by using the TheraScreen assay or laser microdissection. These results indicate that, in the case of post-radiochemotherapy surgical specimens of colorectal cancer with low tumor cellularity, pre-treatment biopsies might represent the most cost-effective option for reliable KRAS genotyping. The use of more sensitive assays, such as allelespecific PCR or laser microdissection, can be envisaged but with higher costs and longer delays.

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“…However, it is time-consuming and labor-intensive and yields less DNA than manual macrodissection [31]. …”

Section: Kras and Braf Mutations: Description And Detection Methodsmentioning

confidence: 99%

The Long and Winding Road to Useful Predictive Factors for Anti-EGFR Therapy in Metastatic Colorectal Carcinoma: The KRAS/BRAF Pathway

et al. 2009

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Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR) have improved outcomes for patients with metastatic colorectal carcinoma. Among patients not carrying activating mutations in the KRAS gene, only a limited number will experience tumor response to these therapeutic agents. The role of BRAF mutations in determining resistance to this treatment is emerging through preclinical and clinical studies. Standardization and validation of laboratory mutation analysis is needed to allow an optimal use of anti-EGFR therapies in the management of colorectal carcinoma. Clinical single-arm and randomized studies were conducted both in first-line and refractory settings to evaluate the correlation of KRAS mutational status and efficacy of cetuximab and panitumumab. The main trials on first-line regiments are CRYSTAL, which is looking at FOLFIRI (folinic acid, fluorouracil, irinotecan) + cetuximab, and OPUS, which is evaluating FOLFOX (folinic acid, fluorouracil, oxaliplatin) + cetuximab. The results of these trials have induced the European Medicines Agency to apply restrictions to its approval of cetuximab and panitumumab for use in metastatic colorectal cancer patients with wild-type KRAS tumors. However, the absence of KRAS mutations is not sufficient to assure clinical response to cetuximab and panitumumab. We need to discover further molecular biomarkers of impairment in this or other signaling pathways to identify responders more specifically. Preclinical rationale is available for combined therapies, which simultaneously target EGFR and the RAS/RAF/MAPK signaling pathways for metastatic colorectal cancer.

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Laser pressure catapulting (LPC): Optimization LPC‐system and genotyping of colorectal carcinomas

Cited by 21 publications

References 17 publications

Cyclin A2 Protein Overexpression Is Not Caused by Gene Amplification in Colon Cancer

Cyclin A2 Protein Overexpression Is Not Caused by Gene Amplification in Colon Cancer

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

The Long and Winding Road to Useful Predictive Factors for Anti-EGFR Therapy in Metastatic Colorectal Carcinoma: The KRAS/BRAF Pathway

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