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Posts Tagged ‘immunohistochemistry’


Prognostic biomarker for NSCLC and Cancer Metastasis

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Membranous CD24 expression as detected by the monoclonal antibody SWA11 is a prognostic marker in non-small cell lung cancer patients

Michael MajoresAnne SchindlerAngela FuchsJohannes SteinLukas HeukampPeter Altevogt and Glen Kristiansen

BMC Clinical Pathology201515:19   http://dx.doi.org:/10.1186/s12907-015-0019-z

Background    Lung cancer is one of the most common malignant neoplasms worldwide and has a high mortality rate. To enable individualized therapy regimens, a better understanding of the molecular tumor biology has still to be elucidated. The expression of the cell surface protein CD24 has already been claimed to be associated with shorter patient survival in non-small cell lung cancer (NSCLC), however, the prognostic value and applicability of CD24 immunostaining in paraffin embedded tissue specimens has been questioned due to the recent acknowledgement of restricted epitope specificity of the commonly used antibody SN3b.   Methods    A cohort of 137 primary NSCLC cases was immunostained with a novel CD24 antibody (clone SWA11), which specifically recognizes the CD24 protein core and the resulting expression data were compared with expression profiles based on the monoclonal antibody SN3b. Furthermore, expression data were correlated to clinico-pathological parameters. Univariate and multivariate survival analyses were conducted with Kaplan Meier estimates and Cox regression, respectively. Results    CD24 positivity was found in 34 % resp. 21 % (SN3b) of NSCLC with a membranous and/or cytoplasmic staining pattern. Kaplan-Meier analyses revealed that membranous, but not cytoplasmic CD24 expression (clone SWA11) was associated with lympho-nodular spread and shorter overall survival times (both p < 0.05). CD24 expression established by SN3b antibodies did not reveal significant clinicopathological correlations with overall survival, neither for cytoplasmic nor membranous CD24 staining.  Conclusions    Membranous CD24 immunoreactivity, as detected with antibody clone SWA11 may serve as a prognostic factor for lymphonodular spread and poorer overall survival. Furthermore, these results corroborate the importance of a careful distinction between membranous and cytoplasmic localisation, if CD24 is to be considered as a potential prognostic biomarker.

 

Lung cancer is a major cause of carcinoma related death, being responsible for 17.8 % of all cancer deaths and accounting for more than a million deaths worldwide per year [1]. Despite intense studies to improve therapy options, its prognosis has remained poor with a 5-year overall survival rate of less than 15 % [2].

In the past decade, the largest subgroup of lung cancer, i.e. non-small cell lung cancer (NSCLC), has been subjected to exerted research for a better understanding of the underlying molecular biology of lung cancer. More than ten years ago, CD24 has already been suggested as a novel and promising biomarker for carcinoma progression in NSCLC [3] and several groups have confirmed this finding on protein and transcript level [2, 4]. CD24 is a highly glycosylated protein, that binds to the cell surface through a GPI (glycosyl-phosphatidylinositol)-anchor and functions as a cell adhesion molecule and is involved in cell-cell-interaction via its P-selectin binding site [5]. CD24 has been found to be expressed by pre-B-lymphocytes [5]. It is assumed that CD24-positive cells can attach more easily to platelets and activated endothelial cells [6, 7]. Notably, CD24 has also been observed in many human carcinomas, such as ovarian cancer, renal cell cancer, breast cancer and NSCLC [3, 812]. In epithelial ovarian cancer high scores of cytoplasmic CD24 were highly predictive of shorter patient survival times (mean 97.8 vs. 36.5 months), whereas membranous CD24 expression seemed to have no influence on survival times. Interestingly, CD24 positivity (membranous or cytoplasmic) of prostate cancer samples was significantly associated to younger patient age and higher pT stages and a higher 3-year prostate-specific antigen (PSA) relapse rate compared with CD24-negative tumours.

In patients with gallbladder carcinoma, tumors with up-regulation of CD24 revealed lymph node metastasis and lymphovascular invasion more frequently. Moreover, up-regulation of CD24 tended to show deeper invasion depth and higher TNM stage [13]. Together, these findings support CD24 as a prognostic marker for carcinoma progression and poorer survival.

Despite these intriguing findings, major concerns regarding a lack of epitope specificity of the commonly used monoclonal antibody SN3b have been raised [14]. Recent findings indicate that the mAb (monoclonal antibody) SN3b does not bind to the protein core itself, but binds to a glycan structure that decorates the CD24 molecule. On the one hand, this motif is not present on all forms of CD24 and—on the other hand—it can be present in other epitopes irrespective of CD24 [14]. These limitations underline the need for more specific CD24 antibodies, such as the mAb SWA11 antibody that has been suggested to be more specific as it binds to the protein core [14].

As CD24 is a promising biomarker for the risk assessment of disease progression, the goal of the present study was to investigate CD24 expression in NSCLC using the novel, more specific monoclonal antibody (mAb) SWA11. Special emphasis was put on the comparison of SN3b- and SWA11-mediated CD24 detection regarding a) the subcellular distribution of CD24 expression (i.e. membranous versus cytoplasmic expression) and b) its correlation with various clinicopathological features including patient survival times.

Table 1

Clinicopathological characteristics of the NSCLC cohort

  AC SCC
N (%) N (%)
Tumour stage (pT)
1 29 (21.2 %) 5 (3.6)
2 51 (37.2 %) 23 (16.8 %)
3 6 (4.4 %) 6 (4.4 %)
4 1 (0.7 %) 0 (0 %)
Nodal Status (pN) 0 37 (27.0 %) 15 (10.9 %)
1 15 (10.9 %) 9 (6.6 %)
2 14 (10.2 %) 3 (2.2 %)
3 1 (0.7 %) 0 (0.0 %)
Grading (G) 1 5 (3.6 %) 0 (0.0 %)
2 41 (29.9 %) 16 (11.6 %)
3 44 (32.1 %) 17 (12.4 %)
Mean age at surgery 64,2 64,56
(median age) (65) (67)
Sex (m:w) 68:34 30:5
Median OS (months) 52 24
(SD; 95 % CI [months]) (±23.7; 5.5– 98.5) (± 12.8;0.0– 49.0)

 

Immunohistochemical detection of CD24 expression using clone SWA11 and SN3b

Using the mAb SWA11, 47 of 137 (34.3 %) NSCLC revealed CD24 expression (either cytoplasmic or membranous) (Table 2). CD24 expression was observed more frequently in adenocarcinomas (AC) than in squamous cell carcinomas (SCC). In AC cytoplasmic expression was observed more frequently than membranous expression. In SCC, both cyptoplasmic and membranous expression was rare. Normal lung parenchyma (i.e. alveolar surface cells) showed no expression of CD24. Bronchial epithelium showed a strong membranous and cytoplasmic staining of the brush border (Fig. 1).

Table 2

Cytoplasmic and membranous expression of CD24

SWA11 (mAb clone) SN3b (mAB clone)
  AC SCC   AC SCC
Cytoplasmic N (%) N (%) Cytoplasmic N (%) N (%)
0 45 (32.6 %) 19 (13.8 %) 0 76 (55.1 %) 31 (22.5 %)
1 22 (15.9 %) 8 (5.8 %) 1 12 (8.7 %) 1 (0.7 %)
2 17 (12.3 %) 4 (2.9 %) 2 7 (5.1 %) 2 (1.4 %)
3 18 (13.0 %) 4 (2.9 %) 3 1 (0.7 %) 0 (0 %)
AC SCC AC SCC
Membranous N (%) N (%) Membranous N (%) N (%)
0 68 (49.3 %) 21 (15.2 %) 0 64 (46.4 %) 30 (21.7 %)
1 21 (15.2 %) 5 (3.6 %) 1 10 (7.2 %) 2 (1.4 %)
2 8 (5.8 %) 4 (2.9 %) 2 12 (8.7 %) 2 1.4 %)
3 5 (3.6 %) 5 (3.6 %) 3 10 (7.2 %) 0 (0 %)

Staining intensities are determined as follows:

0: negative or equivocal, 1: weak, 2: moderate and 3: strong CD24 staining

 

https://static-content.springer.com/image/art%3A10.1186%2Fs12907-015-0019-z/MediaObjects/12907_2015_19_Fig1_HTML.gif

Fig 1

The immunohistochemical characterization reveals membranous and/or cytoplasmic CD24 (mAb SWA11) expression. Strong cytoplasmic CD24 expression is found in a proportion of both AC (a) and SCC (b, d) specimens. Membranous CD24 expression can be pronounced with only scant or even absent cytoplasmic staining as shown in the AC (c). Also, both membranous and cytoplasmic CD24 detection can be found in some instances (d), the insert is showing the corresponding squamous carcinoma in-situ with membranous staining. Simultaneous membranous and cytoplasmic CD24 expression is also found in AC specimens (e, f). In normal tissue, alveolar epithelial cells do not express CD24 (g), whereas CD24 staining is found at the apical cell membrane of bronchial respiratory epithelia (h)

Using the mAb SN3b, 29 of 137 (21.2 %) NSCLC revealed CD24 expression (either cytoplasmic or membranous) (Table 2). As above, CD24 expression was observed more frequently in adenocarcinomas (AC) than in squamous cell carcinomas (SCC). However, in contrast to mAb SWA11 cytoplasmic expression was observed less frequently than membranous expression in AC. In SCC, both cytoplasmic and membranous expression was rare. Normal lung parenchyma (i.e. alveolar surface cells) showed a distinct membranous immunoreactivity. Bronchial epithelium revealed both membranous and cytoplasmic staining of CD24.

Correlation between SWA11 and SN3b: As SWA11 and SN3b detect different epitopes, we evaluated the correlation of the immunohistochemical staining patterns. Of 132 NSCLC specimens with matched expression data, only 9 specimens (6.8 %) revealed a concordant CD24 expression. Of these cases, 4 cases revealed a concordant cytoplasmic staining and another 5 cases revealed a concordant membranous CD24 expression. Statistically, no significant correlation between the two mAb could be observed (cc = −0.63, p = 0.470; Fisher’s exact test p = 0.665). The correlation of cytoplasmic and membranous expression (for each antibody) was as follows: cc = 0.475 (p < 0.05) for SWA11 (n = 108) and cc = 0.140 (p = 0.11) for SN3b (n = 103).

Survival analyses

Recent studies indicate that CD24 expression is associated with tumor progression and poorer survival rates. Therefore, we performed follow up analyses with a special emphasis on 1) the prognostic value of mAb SWA11 in dependence on subcellular staining characteristics and 2) the prognostic values of different clinicopathological parameters:

Prognostic value of CD24 in Kaplan Meier Analyses

Only membranous CD24 (SWA11) staining revealed significantly poorer survival rates (median overall survival 21 vs. 52 months; p = 0.005) as illustrated in Fig. 2. In contrast, cytoplasmic CD24 (SWA11) staining did not affect the survival rates (median OS 34 vs. 35 months; p = 0.884) (Table 3). When stratifying the cohort into SCC (n = 35) and AC (n = 102) in Kaplan Meier analyses, membranous CD24 (SWA11) expression did not affect patients’ survival, neither in SCC (p = 0.243) nor AC (p = 0.135) (Table 3), probably due to the small number of observations (Fisher exact test: p > 0.05). After stratification for AC subtypes, membranous CD24 expression (SWA11) showed a tendency towards an association with poorer survival in acinar subtype AC, but failed significance (p = 0.328).
https://static-content.springer.com/image/art%3A10.1186%2Fs12907-015-0019-z/MediaObjects/12907_2015_19_Fig2_HTML.gif

Fig 2

Survival analysis. Kaplan-Meier curves according to SWA11 expression. Cases with moderate to strong expression were bundled in a ‘high expression’ and cases with negative or weak expression in a ‘low expression’ group. Membranous expression of CD24 detected by SWA11 proved to be an independent marker for shorter survival times in NSCLC (p = 0.005)

Table 3

Univariate survival analysis

SWA11 No. of cases Mean survival time Median survival time p-value
(months +/− s.e.) (months +/− s.e.)
Mem CD24
Negative 76 84.833 +/− 10.395 52.000 +/− 27.030 0.005
Positive 16 27.925 +/− 6.379 21.000 +/− 4.000
Cyto CD24
Negative 66 75.209 +/− 10.577 35.000 +/− 12.422 0.884
Positive 26 60.540 +/− 11.551 34.000 +/− 12.196
Total CD24
Negative 64 76.972 +/− 10.841 35.000 +/− 13.726 0.633
Positive 28 57.535 +/− 10.895 34.000 +/− 9.303
SCC
Mem CD24 negative 16 52.063 +/− 14.668 16.000 +/− 16.000 0.243
Mem CD24 positive 7 21.571 +/− 7.201 24.000 +/− 23.568
AC
Mem CD24 negative 59 88.953 +/− 11.631 56.000 +/− 22.885 0.135
Mem CD24 positive 8 39.167 +/− 11.674 21.000 +/− 8.485
pN0 31 103.641 +/− 14.940 93.000 +/− 28.224 0.012
pN1+ 30 54.911 +/− 10.646 26.000 +/− 0.983

 

…..

Univariate survival analysis according to the Cox regression model (mAb SWA11)

  Beta HR (hazard ratio) 95 % CI of HR P-value
SWA11 mem all 0.856 2.353 1.268–4.364 0.007
pN 0.963 2.620 1.389–4.943 0.003
pT 0.844 2.325 1.279–4.224 0.006
Tumour type 0.975 2.651 1.999–3.517 0.000

Table 5

Multivariate survival analysis according to the Cox regression model (mAb SWA11)

  Beta HR (hazard ratio) 95 % CI of HR P-value
SWA11 mem all 0.944 2.571 1.211–5.458 0.014
pN 0.737 2.091 1.087–4.021 0.027
pT 0.587 1.799 0.755–4.283 0.185

 

…..

In the present study, we have analyzed immunohistochemical staining characteristics and the prognostic value of CD24 expression in NSCLC with a special emphasis on the comparison of the CD24 antibodies SWA11 and SN3b. The most important result of our study is that the prognostic relevance of CD24 is critically dependent on the careful consideration of sub-cellular compartments and the epitope specificity of the antibody used.

Overall, about one third of the NSCLC cohort revealed a significant CD24 expression (either cytoplasmic or membranous). These results are in line with the findings of other studies. In another NSCLC cohort, CD24 (SN3b) expression was found in 33 % of the samples (87 of 267 cases) [2]. Consistent with those results, we have found similar rates of high CD24 expression levels (35 % of the cases) for SWA11. Originally, we would have expected lower rates than those found by Lee et al, as they used the antibody SN3b, that also recognizes yet unidentified other glycoproteins next to CD24. Furthermore, they used whole mount sections instead of tissue microarrays. A possible explanation for rather equal detection rates would be the fact that it has been demonstrated that the epitope recognized by SN3b is indeed present in CD24, but is not found in all glycoforms of CD24 [14]. In contrast to the commonly used mAb SN3b, mAb SWA11 binds to the protein core of CD24 and does not depict other glycan moieties next to CD24. The protein core of CD24 is linear, consisting of the amino acid sequence leucine-proline-alanine (LAP) next to a glycosyl-phosphatidylinositol anchor [15].

CD24 expression has been associated with disease progression and cancer-related death in the majority of malignant tumors [2, 3, 16, 17], although a caveat to these data is that most of these studies are based on the supposedly less specific CD24 clone SN3b. Lee et al demonstrated a significant association between CD24-high expression (SN3b) and shorter patient survival times. Furthermore, Lee and colleagues and ourselves in former studies referred the results to cytoplasmic CD24 expression [2, 3].

Switching Off Cancers’ Ability to Spread

http://www.technologynetworks.com/rnai/news.aspx?ID=189704

A key molecule in breast and lung cancer cells can help switch off the cancers’ ability to spread around the body.

The findings by researchers at Imperial College London, published in the journal EMBO Reports, may help scientists develop treatments that prevent cancer travelling around the body – or produce some kind of test that allows doctors to gauge how likely a cancer is to spread. During tumour growth, cancer cells can break off and travel in the bloodstream or lymph system to other parts of the body, in a process called metastasis.

Patients whose cancers spread tend to have a worse prognosis, explains Professor Justin Stebbing, senior author of the study from the Department of Surgery and Cancer at Imperial: “The ability of a cancer to spread around the body has a large impact on a patient’s survival. However, at the moment we are still in the dark about why some cancers spread around the body – while others stay in one place. This study has given important insights into this process.”

The researchers were looking at breast and lung cancer cells and they found that a protein called MARK4 enables the cells to break free and move around to other parts of the body, such as the brain and liver. Although scientist are still unsure how it does this, one theory is it affects the cell’s internal scaffolding, enabling it to move more easily around the body. The team found that a molecule called miR-515-5p helps to silence, or switch off, the gene that produces MARK4.

In the study, the team used human breast cancer and lung cancer cells to show that the miR-515-5p molecule silences the gene MARK4. They then confirmed this in mouse models, which showed that increasing the amount of miR-515-5p prevents the spread of cancer cells. The findings also revealed that the silencer molecule was found in lower levels in human tumours that had spread around the body. The team then also established that patients with breast and lung cancers whose tumours had low amounts of these silencer molecules – or high amounts of MARK4 – had lower survival rates.

Researchers are now investigating whether either the MARK4 gene or the silencer molecule could be targeted with drugs. They are also investigating whether these molecules could be used to develop a test to indicate whether a patient’s cancer is likely to spread. Professor Stebbing said: “In our work we have shown that this silencer molecule is important in the spread of cancer. This is very early stage research, so we now need more studies to find out more about this molecule, and if it is present in other types of cancer.”

Dr Olivier Pardo, lead author of the paper, also from the Department of Surgery and Cancer at Imperial, added: “Our work also identified that MARK4 enables breast and lung cancer cells to both divide and invade other parts of the body. These findings could have profound implications for treating breast and lung cancers, two of the biggest cancer killers worldwide.” The study was supported by the NIHR Imperial Biomedical Research Centre, the Medical Research Council, Action Against Cancer and the Cancer Treatment and Research Trust.

 

‘Silencer molecules’ switch off cancer’s ability to spread around body

by Kate Wighton

main image

Scientists have revealed that a key molecule in breast and lung cancer cells can help switch off the cancers’ ability to spread around the body

The findings by researchers at Imperial College London, published in the journal EMBO Reports, may help scientists develop treatments that prevent cancer travelling around the body – or produce some kind of test that allows doctors to gauge how likely a cancer is to spread.

During tumour growth, cancer cells can break off and travel in the bloodstream or lymph system to other parts of the body, in a process called metastasis.

Patients whose cancers spread tend to have a worse prognosis, explains Professor Justin Stebbing, senior author of the study from the Department of Surgery and Cancer at Imperial: “The ability of a cancer to spread around the body has a large impact on a patient’s survival. However, at the moment we are still in the dark about why some cancers spread around the body – while others stay in one place. This study has given important insights into this process.”

The researchers were looking at breast and lung cancer cells and they found that a protein called MARK4 enables the cells to break free and move around to other parts of the body, such as the brain and liver. Although scientist are still unsure how it does this, one theory is it affects the cell’s internal scaffolding, enabling it to move more easily around the body.

 

miR‐515‐5p controls cancer cell migration through MARK4 regulation

Olivier E Pardo, Leandro Castellano, Catriona E Munro, Yili Hu, Francesco Mauri,Jonathan Krell, Romain Lara, Filipa G Pinho, Thameenah Choudhury, Adam EFrampton, Loredana Pellegrino, Dmitry Pshezhetskiy, Yulan Wang, JonathanWaxman, Michael J Seckl, Justin Stebbing    

EMBO reports http://embor.embopress.org/content/early/2016/02/10/embr.201540970     http://dx.doi.org:/
Here, we show that miR‐515‐5p inhibits cancer cell migration and metastasis. RNA‐seq analyses of both oestrogen receptor receptor‐positive and receptor‐negative breast cancer cells overexpressing miR‐515‐5p reveal down‐regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR‐515‐5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock‐down mimics the effect of miR‐515‐5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR‐515‐5p, suggesting miR‐515‐5p mediates this process through MARK4 down‐regulation. Furthermore, miR‐515‐5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR‐515‐5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR‐515‐5p and low MARK4 expression correlate with increased breast and lung cancer patients’ survival, respectively. Taken together, these data demonstrate the importance of miR‐515‐5p/MARK4 regulation in cell migration and metastasis across two common cancers.
Embedded Image

miR‐515‐5p inhibits cancer progression, cell migration and metastasis through its direct target MARK4, a regulator of the cytoskeleton and cell motility. Moreover, reduced miR‐515‐5p and increased MARK4 levels in metastatic lung and breast cancer correlate with poor patient prognosis.

  • MARK4 down‐regulation promotes microtubule polymerisation.

  • Increased cell spreading downstream of miR‐515‐5p overexpression or MARK4 silencing hinders cell motility and invasiveness.

  • miR‐515‐5p overexpression or MARK4 silencing prevent organ colonisation by circulating tumour cells.

  • MARK4 inhibitors may represent novel therapeutic agents to control cancer dissemination.breasat cancer

 

Liquid Biopsy for NSCLC

http://www.technologynetworks.com/Diagnostics/news.aspx?ID=190276

‘Liquid biopsy’ blood test accurately detects key genetic mutations in most common form of lung cancer, study finds.

A simple blood test can rapidly and accurately detect mutations in two key genes in non-small cell lung tumors, researchers at Dana-Farber Cancer Institute and other institutions report in a new study – demonstrating the test’s potential as a clinical tool for identifying patients who can benefit from drugs targeting those mutations.

The test, known as a liquid biopsy, proved so reliable in the study that Dana-Farber/Brigham and Women’s Cancer Center (DF/BWCC) expects to offer it soon to all patients with non-small cell lung cancer (NSCLC), either at the time of first diagnosis or of relapse following previous treatment.

NSCLC is the most common form of lung cancer, diagnosed in more than 200,000 people in the United States each year, according to the American Cancer Society. An estimated 30 percent of NSCLC patients have mutations in either of the genes included in the study, and can often be treated with targeted therapies. The study is being published online today by the journal JAMA Oncology.

The liquid biopsy tested in the study – technically known as rapid plasma genotyping – involves taking a test tube-full of blood, which contains free-floating DNA from cancer cells, and analyzing that DNA for mutations or other abnormalities. (When tumor cells die, their DNA spills into the bloodstream, where it’s known as cell-free DNA.) The technique, which provides a “snapshot” of key genetic irregularities in a tumor, is a common tool in research for probing the molecular make-up of different kinds of cancers.

“We see plasma genotyping as having enormous potential as a clinical test, or assay – a rapid, noninvasive way of screening a cancer for common genetic fingerprints, while avoiding the challenges of traditional invasive biopsies,” said the senior author of the study, Geoffrey Oxnard, MD, thoracic oncologist and lung cancer researcher at Dana-Farber and Brigham and Women’s Hospital. “Our study was the first to demonstrate prospectively that a liquid biopsy technique can be a practical tool for making treatment decisions in cancer patients. The trial was such a success that we are transitioning the assay into a clinical test for lung cancer patients at DF/BWCC.”

The study involved 180 patients with NSCLC, 120 of whom were newly diagnosed, and 60 of whom had become resistant to a previous treatment, allowing the disease to recur. Participants’ cell-free DNA was tested for mutations in the EGFR and KRAS genes, and for a separate mutation in EGFR that allows tumor cells to become resistant to front-line targeted drugs. The test was performed with a technique known as droplet digital polymerase chain reaction (ddPCR), which counts the individual letters of the genetic code in cell-free DNA to determine if specific mutations are present. Each participant also underwent a conventional tissue biopsy to test for the same mutations. The results of the liquid biopsies were then compared to those of the tissue biopsies.

The data showed that liquid biopsies returned results much more quickly. The median turnaround time for liquid biopsies was three days, compared to 12 days for tissue biopsies in newly diagnosed patients and 27 days in drug-resistant patients.

Liquid biopsy was also found to be highly accurate. In newly diagnosed patients, the “predictive value” of plasma ddPCR was 100 percent for the primary EGFR mutation and the KRAS mutation – meaning that a patient who tested positive for either mutation was certain to have that mutation in his or her tumor. For patients with the EGFR resistance mutation, the predictive value of the ddPCR test was 79 percent, suggesting the blood test was able to find additional cases with the mutation that were missed using standard biopsies.

“In some patients with the EGFR resistance mutation, ddPCR detected mutations missed by standard tissue biopsy,” Oxnard remarked. “A resistant tumor is inherently made up of multiple subsets of cells, some of which carry different patterns of genetic mutations. A single biopsy is only analyzing a single part of the tumor, and may miss a mutation present elsewhere in the body. A liquid biopsy, in contrast, may better reflect the distribution of mutations in the tumor as a whole.”

When ddPCR failed to detect these mutations, the cause was less clear-cut, Oxnard says. It could indicate that the tumor cells don’t carry the mutations or, alternatively, that the tumor isn’t shedding its DNA into the bloodstream. This discrepancy between the test results and the presence of mutations was less common in patients whose cancer had metastasized to multiple sites in the body, researchers found.

The ddPCR-based test, or assay, was piloted and optimized for patients at the Translational Resarch lab of the Belfer Center for Applied Cancer Science at Dana-Farber. It was then validated for clinical use at Dana-Farber’s Lowe Center for Thoracic Oncology.

An advantage of this form of liquid biopsy is that it can help doctors quickly determine whether a patient is responding to therapy. Fifty participants in the study had repeat testing done after starting treatment for their cancer. “Those whose blood tests showed a disappearance of the mutations within two weeks were more likely to stay on the treatment than patients who didn’t see such a reduction,” said the study’s lead author, Adrian Sacher, MD, of Dana-Farber and Brigham and Women’s Hospital.

And because tumors are constantly evolving and acquiring additional mutations, repeated liquid biopsies can provide early detection of a new mutation – such as the EGFR resistance mutation – that can potentially be treated with targeted agents.

“The study data are compelling,” said DF/BWCC pathologist Lynette Sholl, MD, explaining the center’s decision to begin offering ddPCR-based liquid biopsy to all lung cancer patients. “We validated the authors’ findings by cross-comparing results from liquid and tissue biopsies in 34 NSCLC patients. To work as a real-world clinical test, liquid biopsy needs to provide reliable, accurate data and be logistically practical. That’s what we’ve seen with the ddPCR-based blood test.

“The test has great utility both for patients newly diagnosed with NSCLC and for those with a recurrence of the disease,” she continued. “It’s fast, it’s quantitative (it indicates the amount of mutant DNA in a sample), and it can be readily employed at a cancer treatment center.”

The co-authors of the study are Cloud Paweletz, PhD, Allison O’Connell, BSc, and Nora Feeney, BSc, of the Belfer Center for Applied Cancer Science at Dana-Farber; Ryan S. Alden BSc, and Stacy L. Mach BA, of Dana-Farber; Suzanne E. Dahlberg, PhD, of Dana-Farber and Harvard T.H. Chan School of Public Health; and Pasi A. Jänne, MD, PhD, of Dana-Farber, the Belfer Center, and Brigham and Women’s Hospital.

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Reporter and Curator: Ritu Saxena, PhD

Magnetic Resonance Imaging (MRI) is increasingly used in clinical diagnostics, for a rapidly growing number of indications. The MRI technique is non-invasive and can provide information on the anatomy, function and metabolism of tissues in vivo (Strijkers GJ, et al, Anticancer Agents Med Chem, May 2007;7(3):291-305). Basic contrast in the MRI image scans is as a result of contrast generated by differences in the relaxation times between different regions. Since the intrinsic contrast generated between regions is limited to allow clear and specific diagnosis, MRI contrast agents administered intravenously are increasingly being used to alter image contrast.

Gadoxetic acid, a gadolinium-based compound, is a recently developed hepatobiliary-specific contrast material for MRI that has high sensitivity in the detection of malignant liver tumors. Its salt, gadoxetate disodium, is marketed as Primovist in Europe and Eovist in the United States by Bayer HealthCare Pharmaceuticals. Gadoxetic acid is taken up by hepatocytes and then excreted into the bile ducts (Schuhmann-Giampieri G, et al, Radiology, Apr 1992;183(1):59-64). Therefore, hepatic focal lesions without normal hepatobiliary function are depicted as hypointense areas compared with the well-enhanced hyperintense background liver in the hepatobiliary phase of gadoxetic acid–enhanced MR imaging. In addition, gadoxetic acid can be used in the same way as gadopentetate dimeglumine to evaluate the hemodynamics of hepatic lesions in the dynamic phase after an intravenous bolus injection (Kitao A, et al, Radiology, Sep 2010;256(3):817-26).

Recently, researchers from Kanazawa University Graduate School of Medical Science, (Kanazawa, Japan) analyzed the correlation among biologic features, tumor marker production, and signal intensity at gadoxetic acid-enhanced MR imaging in hepatocellular carcinomas (HCCs). The findings were published in Radiology journal. The research was supported in part by a Grant-in-Aid for Scientific Research (21591549) from the Ministry of Education, Culture, Sports, Science and Technology; and by Health and Labor Sciences Research Grants for “Development of novel molecular markers and imaging modalities for earlier diagnosis of hepatocellular carcinoma.”

Research significance: HCC is the most frequent primary malignant tumor of liver and is the third most common cause of cancer death worldwide. It is the most Hepatocellular.

The accurate detection and characterization of HCC focal lesions is crucial for improving prognosis of patients with HCC.

Research problem: Gadoxetic acid–enhanced MR imaging is highly accurate for diagnosing HCC lesions. As discussed earlier, in this imaging process, hepatic focal lesions without normal hepatobiliary are hypointense as compared with the well-enhanced hyperintense background liver. However, approximately 6%–15% of hypervascular HCCs demonstrate isointensity or hyperintensity (Kitao A, et al, Eur Radiol, Oct 2011;21(10):2056-66).

Hypothesis: The reason for hyperintensity in some HCC lesions was previously shown to be due to overexpression of organic anion transporting polypeptide 8 (OATP8) (Kitao A, et al, Radiology, Sep 2010;256(3):817-26). The authors speculated that there might be a correlation of the tumor marker production and signal intensity (SI) on hepatobiliary phase images, which would reflect distinct genomic and proteomic expression of HCC. Thus, authors stated that “the purpose of this study was to analyze the correlation among the pathologic and biologic features, tumor marker production, with signal intensity (SI) on hepatobiliary phase gadoxetic acid–enhanced MR images of HCC” (Kitao A, et al, Radiology, Dec 2012;265(3):780-9).

Experimental design: From April 2008 to September 2011, 180 surgically resected HCCs in 180 patients (age, 65.0 years ± 10.3 [range, 34–83 years]; 138 men, 42 women) were classified as either hypointense (n = 158) or hyperintense (n = 22) compared with the signal intensity of the background liver on hepatobiliary phase gadoxetic acid–enhanced MR images (Abstract of the study).

Pathologic features were analyzed.

Serum analysis and immunohistochemical staining was performed and following were compared:

  1. Alpha fetoprotein (AFP) – is a main tumor marker of HCCs. AFP is the most abundant plasma protein found in the human fetus and plasma levels decrease rapidly after birth. A level above 500 nanograms/milliliter of AFP in adults can be indicative of hepatocellular carcinoma, germ cell tumors, and metastatic cancers of the liver.
  2. Absence of protein induced by vitamin K or antagonist-II (PIVKA-II) – is a clinically important serum tumor marker. PIVKAII is an incomplete coagulation factor prothrombin II whose production is related to the absence of vitamin K or the presence of the antagonist of vitamin K, which is the cofactor of g carboxylase that converts precursor into prothrombin.

Serum levels of both AFP and PIVKA-II correlate with HCC malignancy and prognosis (Miyaaki H, et al, J Gastroenterol, Dec 2007;42(12):962-8).

Results: The hyperintense HCCs showed significantly higher differentiation grade than the hypointense HCCs (P = .028). There was a significant difference in the proliferation pattern between the hypointense and hyperintense HCCs (P < .001) and the hyperintense HCCs showed a significantly lower rate of portal vein invasion than that of hypointense HCCs (P = .039). The serum levels of tumor markers AFP, AFP-L3, and PIVKA-II were significantly lower in the patients with hyperintense HCCs than in those with

hypointense HCCs (P = .003, .004, and .026). In addition, immunohistochemical analysis revealed that the expression of FP and PIVKA-II was lower in hyperintense than in hypointense HCCs (both P < .001). Also, hyperintense HCCs showed lower recurrence rate than hypointense HCCs (P = .039).

Conclusion: Variation was observed within differently stained lesions of HCC in the hepatobiliary phase gadoxetic acid–enhanced MR images as evident in tumor marker expression, proliferation pattern, differentiation grade, immunohistochemical analysis and recurrence.  The results lead to the hypothesis that hyperintense HCCs in the hepatobiliary phase gadoxetic acid–enhanced MR images might represent a particular type of HCC that is hypervascular and biologically less aggressive as compared to hypovascular HCCs. Interestingly, this research is another great example where tumor heterogeneity has been brought to light (similar to genetic heterogeneity in triple negative breast cancer deciphered by Lehmann BD, et al, 2011). The heterogeneity might be the basis of answers to why a particular therapy fails in a certain tumor type and fortifying evidence for appropriate analysis of the tumor for obtaining the desired tumor response from a particular drug.

Reference:

Kitao A, et al, Radiology, Dec 2012;265(3):780-9

Strijkers GJ, et al, Anticancer Agents Med Chem, May 2007;7(3):291-305

Schuhmann-Giampieri G, et al, Radiology, Apr 1992;183(1):59-64

Kitao A, et al, Radiology, Sep 2010;256(3):817-26

Kitao A, et al, Eur Radiol, Oct 2011;21(10):2056-66

Kitao A, et al, Radiology, Sep 2010;256(3):817-26

Miyaaki H, et al, J Gastroenterol, Dec 2007;42(12):962-8

Lehmann BD, et al, J Clin Invest, 2011;121(7):2750–2767

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Author and Reporter: Ritu Saxena, Ph.D.  

Mitochondria is an important cell organelle that is associated with several key cellular functions as energy production, anabolism, calcium homeostasis and cell programmed death, and any abnormalities occurring in mitochondria would lead to alteration of normal cellular function.

Role of mitochondria in cancer has long been implicated. Post published on September 1, 2012 (https://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/) presents a brief overview of the mechanisms by which mitochondrial defects could be associated with cancer. Different studies on various types of Cancers have tried to determine the mtDNA mutations and the mechanisms involved. An important aspect of cancerous progression is the cancer cell migration and it has been observed that mitochondrial dysfunction is involved in cancer cell migration. However, the molecular mechanism still needs to be deciphered.

A group from Taiwan recently published their findings in the Biochimica et Biophysica Acta journal stating that enhanced β5-integrin expression was involved in promoting cell migration in human gastric cancer cell line as a result of mitochondrial dysfunction.

The authors used human gastric cancer cell line, SC-M1 cells for their studies. The methodology followed was to first create mitochondrial dysfunction in the SC-M1 cells by the use of oxidative phosphorylation inhibitors: oligomycin (Complex V inhibitor) and antimycin A (Complex III inhibitor) thereby inhibiting mitochondrial function. The results indicated that impaired oxidative phosphorylation caused an increase in the intracellular Reactive Oxygen Species (ROS) that lead to an increased cell migration in SC-M1 cells.

Different types of integrin molecules have been implicated in cell migration. Hung et al extracted RNA and protein from SC-M1 cells in order to study the different types of integrins, and observed that the levels of β5-integrin were significantly upregulated in SC-M1 cells.  Simultaneously, the surface expression of the dimer- β5-integrin and αv-integrin, was studied in cancer cells with using FACS. The analysis revealed a higher surface expression of the dimer corresponding to the higher levels of the protein and RNA results of  β5-integrin expression in SC-M1 cells with mitochondrial dysfunction. Infact, a subpopulation of SC-M1 cells that showed higher migration capability (SC-M1-3rd) was observed to harbor a higher lever of β5-integrin expression, correlating β5-integrin expression with cell migration ability. The experiments supported the role of β5-integrin in cell migration in gastric cancer cells.

Finally, authors confirmed the in vitro results in the human gastric cancer samples. Immunohistochemical analysis revealed that β5-integrin was stained positive in around 73% of the cancer samples. Additionally, the higher expression levels of β5-integrin could be correlated with the invasive ability and more aggressive behavior of gastric cancer cells.

Authors stated “our study pinpoints another aspect that links the induction of intracellular ROS level in mitochondrial dysfunction gastric cancer cells with the activation of αvβ5-integrin. Taken together, the induction of β5-integrin is important to gastric cancer metastasis, especially in cancer cells that exhibit mitochondrial dysfunction.”

Thus, blockage of αvβ5-integrin function by antibodies might be tested as a potential therapy for preventing or delaying gastric cancer metastasis, especially in gastric cancers harboring mitochondrial dysfunction.

Sources:

Research article: http://www.ncbi.nlm.nih.gov/pubmed?term=22561002

Related posts: https://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/

https://pharmaceuticalintelligence.com/2012/09/06/clinical-genetics-personalized-medicine-molecular-diagnostics-consumer-targeted-dna-consumer-genetics-conference-cgc-october-3-5-2012-seaport-hotel-boston-ma/

https://pharmaceuticalintelligence.com/2012/08/14/detecting-potential-toxicity-in-mitochondria/

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Author and Reporter: Ritu Saxena, Ph.D.

On June 4, 2012, I authored a post on HBV and HCV-associated Liver Cancer: Important Insights from the Genome https://pharmaceuticalintelligence.com/2012/06/04/hbv-and-hcv-associated-liver-cancer-important-insights-from-the-genome/ reporting about the major role of chromatin remodeling complexes and involvement of both interferon and oxidative stress pathways in hepatocellular malignant proliferation and transformation based on the genes showing recurrent mutations in the observed genes.

In this post, I have discussed the latest research on cyclin B1 and Sec62 expression in PBMCs of HCC patients and how their elevated expression correlates to significantly to negative prognostic value in terms of recurrence-free survival.

Researchers at the Changhai and Gongli Hospital in Shanghai, Military Medical University, People’s Republic of China recently identified the candidate biomarkers for HBV-related HCC recurrence after surgery. The research was published in the June 2012 issue of Molecular Cancer journal. According to the group findings, Cyclin B1 and Sec62 may serve as effective biomarkers and potential therapeutic targets for HBV-related HCC recurrence after surgery.

Research article: Identification of cyclin B1 and Sec62 as biomarkers for recurrence in patients with HBV-related hepatocellular carcinoma after surgical resection. http://www.ncbi.nlm.nih.gov/pubmed/22682366

HCC background and Research Problem: Hepatocellular carcinoma is cancer of the liver. It is different from Metastaticc liver cancer, which starts in another organ (such as the breast or colon) and spreads to the liver. The most frequent factors causing HCC include chronic viral hepatitis (types B and C), alcohol intake and afla- toxin exposure.

In most cases, scarring of the liver referred to as cirrhosis is an important risk factor for HCC. Cirrhosis may be caused by:

http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001325/.

However, patients with hepatitis B or C are at risk for liver cancer, even if they have not developed cirrhosis.

According to the data from International Agency for Research on Cancer, hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, with over a half million deaths per annum. http://www-dep.iarc.fr/

In China, a very high infection rates with HBV have been reported. According to the recent statistics reported by Jemal et al in 2011, HCC cases occurring in China account for 55% of the total cases reported in the world. http://www.ncbi.nlm.nih.gov/pubmed?term=Global%20Cancer%20Statistics%20Jemal

Surgical resection, although provides an opportunity for cure, however, frequent recurrence post surgery has posed a major challenge to longterm survival. Pertinent to their research, authors state “Frequent tumor recurrence after surgery is related to its poor prognosis. Although gene expression signatures have been associated with outcome, the molecular basis of HCC recurrence is not fully understood..”.

Research: To determine the molecular basis of HCC, authors used the Peripheral blood mononuclear cells (PBMCs) to predict the recurrence of HCC after surgery. Use of PBMCs was in contrast to previous studies that used just the liver tissues. PBMCs have the advantage of being easily obtained in the clinical setting. Thus, identification of biomarkers using PBMCs would be a great way to predict the recurrence of HCC post surgery.

A microarray-based gene expression profiling was performed to indentify candidate genes related to HCC recurrence. In all, mRNA derived from 6 HCC cases (3 cases with recurrence and 3 without recurrence) were subjected to genome-wide analysis. Some critical genes were indentified including cyclin B1 (CCNB1), SEC62 homolog (S. cerevisiae) (SEC62), and baculoviral IAP repeat-containing 3 (BIRC3), suggesting that they probably play important roles in the pathogenesis of HCC recurrence. To confirm the results of microarray analysis, the mRNA and protein expressions of these 3 genes were measured in 80 HCC samples from HCC cases and 30 samples from healthy cases. The authors found that the transcriptional and protein expressions of cyclin B1, Sec62, and Birc3 in the PBMCs were significantly higher in HCC samples than those in the non-recurrent and normal samples.

Furthermore, to determine the clinicopathologic significance of cyclin B1, Sec62, and Birc3 in HCC, immunohistochemical analysis from 35 recurrent tissues and 45 nonrecurrent revealed that the protein levels of cyclin B1, Sec62, and Birc3 were substantially higher in the recurrent tissues than those in the non-recurrent samples. Thus, the immunohistochemical results from tissues were consistent with the previous transcriptional and protein results in PBMCs.

Conclusion of study:  The authors discussed that “In recent years, studies on malignant tumors has primarily focused on cell proliferation, migration, and apoptosis. Cyclin B1, Sec62, and Birc3, chosen in this study according to our microarray analysis, likely play important roles in cell proliferation and migration. They can exert a tumor-promoting effect on HCC by regulating cell cycle and protein translocation.” As derived from the statistical methods employed in the research, elevated cyclin B1 and Sec62 expression in PBMCs had a significantly negative prognostic value in terms of recurrence-free survival, which hints the potential use of these molecular markers to predict the risk of tumor recurrence after surgery and to act as therapeutic targets to reduce tumor recurrence and improve clinical therapies.

Thus, these results revealed that cyclin B1 and Sec62 may be candidate biomarkers and potential therapeutic targets for HBV-related HCC recurrence after surgery.

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