From the 5th Annual Scientific Symposium of the Hong Kong Cancer Institute and the 17th Meeting of the International Academy of Tumour marker Oncology, Hong Kong, China, 23-24 March 2000.

Molecular Monitoring of Tumour Response

Prof. PJ Johnson
The Chinese University of Hong Kong
Hong Kong, China.

Conventional assessment of response to cancer therapy assumes that tumour volume is proportional to the viable tumour mass, explained Prof. Johnson. Tumour volume is usually measured by an imaging procedure and the responses are classified according to the criteria in table 1.

However, conventional approaches may not accurately detect the response, since they only relate to gross disease without detecting important subclinical disease. In addition, conventional approaches do not detect tumour cells in the circulation. Molecular markers have the potential to detect subclinical disease.

Principles of Molecular Monitoring


Prof. Johnson gave an example of the management of liver cancer involving tumour shrinkage with chemotherapy prior to resection of the tumour. Interestingly, although a partial response was obtained, the tumour remained visible on computed tomography (CT) scanning. However, on histopathological examination after resection, the tumour tissue was necrosed with no viable tissue remaining, indicating complete pathological remission prior to surgery.

The tumour marker alpha-fetoprotein can be used to verify remission status since it rapidly decreases to zero following a complete pathological response to treatment conversely, progressive disease corresponds with a rise in alpha-fetoprotein. This molecular marker is more accurate than conventional markers.

Leung et al. examined the response to treatment with PIAF (cisplatin, doxorubicin, 5-fluorouracil, interferon-alpha) by conventional criteria and found no complete responses and a small percentage of patients with a partial response (table 2).¹ In reality, the complete and partial response rates were shown to be higher when alpha-fetoprotein was used as a marker. One important effect of inaccurate monitoring is that the development of new drugs may be delayed if they are initially thought to be ineffective.

Intradose injectable gel is based on the principle of directly injecting a liver tumour with a gel and a chemotherapeutic agent. One effect of this method is that although it may kill the tumour, the volume may increase. However, studies have shown that the alpha-fetoprotein levels rapidly decrease to zero after treatment, implying that the tumour seen on CT is necrosed tissue. Several patients in this series have since had liver transplants, with histopathological examination showing the tumour to be completely necrosed. "It is difficult to monitor tumour response accurately using conventional imaging techniques", explained Prof. Johnson. "However, there are more sophisticated techniques that give a correct answer."

Finding an appropriate molecular marker involves 3 possibilities:

• proteomics generating a general method for discovery of tumour specific proteins
  
• aberrant p16 methylation in plasma using specific mutations of tumour DNA
• other 'tumour-associated' products Epstein-Barr virus DNA.

Proteomics


Proteomes are defined as the entire complement of expressed proteins by a genome, cell, or tissue type.² The techniques that have been instrumental to this approach come from high-resolution 2-dimensional polyacrylamide gel electrophoresis that can resolve all serum proteins into separate spots using isoelectric focusing or molecular weight. The proteins may then be identified by mass spectroscopy. Currently 6000 separate proteins within the plasma can be seen.

The principle of detection of tumour markers by proteomics is to perform 2-dimensional isoelectric focusing of both normal and tumour serum. Using computer software, the normal serum is removed from the tumour serum leaving the tumour-specific serum proteins. Identification is then performed by mass spectroscopy and the glycan moiety is similarly identified after hydrazinolysis.

Using this technique, the Rosetta software programme can successfully identify markers that are either over- or under-expressed in a tumour. The significance of the difference between the tumour and healthy serum can be determined as well as the incidence of the marker and the identity of the protein. This method of detecting tumour- specific proteins "shows a lot of promise for the future."

Other Tumour-associated Products


The best example of the use of other tumour-associated products to monitor response is Epstein-Barr virus (EBV) DNA. Nasopharyngeal carcinoma (NPC) is associated with EBV, as are many of the lymphomas in Hong Kong. Using real-time polymerase chain reaction, it is possible to detect EPV DNA in the plasma of patients with NPC. An EBV-positive cell line (Namalwa) can be used for calibration of the assay and genome copies/ml can be measured in the plasma.³ Treatment of NPC by radiotherapy rapidly decreases levels of EBV DNA to within normal limits, suggesting it may be a useful marker.

Interestingly, in a study described by Prof. Johnson, patients with no EBV DNA after radiotherapy for NPC remained free of disease, while their counterparts who retained EBV DNA after treatment either relapsed or had progressive disease.

Importantly, EBV DNA may detect subclinical disease. All tumours start with 1 malignant cell, which multiplies, until it finally becomes clinically detectable at about 1 cm diameter (figure 1). Similarly, treatment shrinks the tumour to an undetectable size, resulting in so-called 'complete remission'. However, clinically undetectable cells may still be present in large numbers. Using sensitive tumour markers, it may be possible to quantify the status of clinically undetectable disease.

Figure 1. Potential period of subclinical disease that may be detected by Epstein-Barr virus in plasma.

Detection of Circulating Tumour Cells


Use of molecular markers to detect circulating tumour cells in the blood is based on the principle that messenger RNA (mRNA) for tissue specific proteins should not be detectable in the blood. If these proteins are detected in the blood, this implies the presence in the circulatory system of cells from that organ, which are likely to be malignant. Detection is by PCR, and the assay may be quantitated by southern blot analysis of a cell line.

Examination of blood before, during and after surgery for liver cancer shows that most patients do not have circulating cells prior to surgery. However, there is a dramatic increase in cells in the circulation during the operation, with all the potential for metastasis.⁴ None of the patients in whom the circulating cells decreased to zero postoperatively developed disease recurrence, although 5 of 7 patients with continued circulating cells developed recurrence. implying that the cells detected in plasma are significant.

Conclusion


It is possible to move from molecules to management using proteins, glycan moiety of glycoprotein, DNA, mRNA, or virus-associated proteins. Whatever level of molecular testing is used, this approach offers more effective management. Use of molecular markers ensures accurate response monitoring, which may have a real impact on therapy. The development of molecular oncology to monitor tumours in conjunction with conventional monitoring may have an impact for patients before gene therapy becomes available.

References


1. Leung TW, Patt YZ, Lau WY, et al. Complete pathological remission is possible with systemic combination chemotherapy for inoperable hepatocellular carcinoma. Clin Cancer Res 1999; 5:1676-1681.
2. Wilkins MR, Pasquali C, Appel RD, et al. From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Biotechnology (NY) 1996;14(1):61-65.
3. Lo YM, Chan LY, Chan AT, et al. Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 1999; 59(21):5452-5455.
4. Wong IH, Lau WY, Leung T, et al. Hematogenous dissemination of hepatocytes and tumor cells after surgical resection of hepatocellular carcinoma: a quantitative analysis. Clin Cancer Res 1999; 5(12):4021-4027.

Multimodality Treatment of NSCLC. [Abstract]

Prof Everett E Vokes
Departments of Medicine and Radiation Oncology
The University of Chicago
USA

The goals for combined modality therapy for intermediate stage non-small cell lung cancer (NSCLC) are increased survival, organ preservation, prevention of second primary tumours, and reversal of premalignancy. Approaches to multimodality treatment include:

• induction chemotherapy followed by surgery and postoperative radiation therapy
  
• concomitant chemoradiotherapy
  
• concomitant chemoradiotherapy followed by surgery

Considerations include histology, resectability, and performance status.

Stage III NSCLC is generally reasonably confined, although many patients do have systemic micrometastases. Therefore, therapy for lung cancer requires treatment of micrometastases as well as aggressive local therapy.

Multimodality Treatment of HCC

Dr Thomas WT Leung
Department of Clinical Oncology
The Chinese University of Hong Kong
Hong Kong, China

Hepatocellular carcinoma (HCC) is aggressive and often resistant to treatment. Surgery is generally the preferred treatment although many patients present at an advanced stage of disease and surgical resection is not always possible. In addition, patients undergoing surgery are at high risk for recurrence.

Only 11% of patients with HCC have an operable tumour at presentation at the Prince of Wales Hospital (figure 1). Most patients present at an advanced stage with poor liver function and are not suited for treatment. However, many patients presenting with 'inoperable' disease because of the tumour size still have good liver function and performance status and are able to undergo non-surgical treatment.

Figure 1. Treatment for hepatocellular carcinoma at the Prince of Wales Hospital.

The goals of multimodality treatment of HCC are 2-fold. For operable stage disease, the addition of pre- or post-operative treatment may reduce recurrence rates, while induction of complete clinical remission is the goal for inoperable disease.

Operable HCC


Dr Leung explained a new approach to adjuvant treatment used at the Prince of Wales Hospital involving radioactive lipiodoliodine 131. After delivery via the hepatic artery, the lipiodol is taken up by the tumour where a sufficiently tumouricidal radiation dose is delivered. When used in conjunction with surgery, the intention is to reduce the amount of microscopic disease in the liver after tumour resection. Patients given this treatment have significantly longer survival times than those patients not receiving adjuvant therapy (p = 0.022). A single dose of adjuvant lipiodol-iodine-131 treatment is safe and effective as a postoperative adjuvant treatment, reducing recurrence rates and increasing disease-free and overall survival.

Inoperable HCC


Multimodality treatment for inoperable HCC usually involves systemic combination chemotherapy followed by surgery. Other treatments include intra-arterial therapy (chemotherapy, chemotherapy with lipiodisation, chemoembolisation) and internal radiotherapy, or a combination of chemotherapy, intra-arterial therapy, and surgery.

The response to the PIAF (cisplatin, doxorubicin, 5- fluorouracil, interferon-alpha) chemotherapy regimen is often sufficient for surgical resection to take place. Lung metastases also respond well to this chemotherapy regimen. So far, approximately 100 patients with HCC have received the PIAF regimen at the Prince of Wales Hospital. 20% have had their tumours rendered operable with a 2-year survival of 100%. Complete pathological remission has been obtained for 50% of patients, and the remainder had > 95% necrosis of the tumour. In addition, internal radiotherapy using yttrium- 90 microspheres can render tumours operable, often with complete pathological remission.

Conclusion


Combined modality treatment can produce dramatic results for patients with HCC. Previously, surgery or single agent chemotherapy provided only a 5% cure rate for HCC. Now, however, with multimodality treatment using lipiodol or combination chemotherapy, the cure rate has increased to approximately 15%, indicating the progress being made in treating this aggressive disease.

Multimodality Treatment of Nasopharyngeal Carcinoma [Abstract]

Dr Anthony TC Chan
Department of Clinical Oncology
The Chinese University of Hong Kong
Hong Kong, China

Nasopharyngeal carcinoma (NPC) has a high incidence in Asia and is distinctive from other head and neck tumours in that it affects a younger age-group and has a histological pattern of more than 95% undifferentiated carcinoma.

Progress is being made in locoregionally advanced disease by using concurrent chemoradiotherapy. High response rates are achieved with platinum-based chemotherapy for metastatic disease. Future management for NPC is likely to include multimodality approaches, perhaps with immunotherapy.